Am J Physiol Lung Cell Mol Physiol
281: L31–L38, 2001.
Antioxidant imbalance in the lungs of cystic fibrosis
transmembrane conductance regulator protein mutant mice
LEONARD W. VELSOR,1 ANNA VAN HEECKEREN,2 AND BRIAN J. DAY1,3
Department of Medicine, National Jewish Medical and Research Center, and 3Department
of Medicine and Pharmaceutical Sciences, University of Colorado Health Sciences Center,
Denver, Colorado 80206; and 2Department of Pediatrics, Case Western Reserve
University, Cleveland, Ohio 44106
1
Received 3 August 2000; accepted in final form 15 February 2001
epithelial lining fluid; glutathione; oxidative stress
(CF) is a lethal autosomal recessive
disorder associated with mutations in the gene encoding for the CF transmembrane conductance regulator
(CFTR) protein. In the general population, CF has an
overall incidence of ⬃1:3,000, with large differences
among ethnic groups (27). CFTR is a 168-kDa integral
membrane protein primarily expressed in the epithelia
of the lung, pancreas, sweat glands, and vas deferens
(27). CFTR couples ATP hydrolysis with the transport
CYSTIC FIBROSIS
Original submission in response to a special call for papers on
“CFTR Trafficking and Signaling in Respiratory Epithelium.”
Address for reprint requests and other correspondence: B. J. Day,
National Jewish Medical and Research Center, 1400 Jackson St.,
Rm. K-706, Denver, CO 80206 (E-mail: [email protected]).
http://www.ajplung.org
of Cl⫺ and possibly other large anions across apical cell
membranes to maintain the composition of secretions
on the epithelial surfaces (26, 27). Although CF has
serious clinical implications for the gastrointestinal
and genital tracts, pulmonary disease is the primary
cause of death in 90% of CF patients (27).
The progressive obstructive lung disease associated with CF is maintained by recurrent episodes of
infection, predominantly by Pseudomonas aeruginosa (10). A current view is that the inflammatory
responses associated with the persistent infection
drive an injury and repair process that leads to
pulmonary fibrosis, airway obstruction, and, ultimately, respiratory failure (20). Tissue injury is a
direct consequence of the oxidative environment created by the inflammatory response. Inflammatory
cell-derived oxidants and proteases react with critical cellular biomolecules (i.e., lipids, DNA, and proteins) and lead to cell necrosis. Chronic repair processes lead to fibrosis and the progressive
deterioration of pulmonary function (20).
In vitro studies suggest that CFTR may be involved
in maintaining the antioxidant homeostasis of the pulmonary epithelial lining fluid (ELF), and a mutation in
CFTR may impair lung antioxidant defenses, thereby
making the CF lung more susceptible to oxidative
stress and fibrosis (16, 25). Analysis of bronchoalveolar
lavage fluid (BALF) from adult CF patients demonstrated that these patients have lower concentrations
of ELF reduced glutathione (GSH) than normal control
subjects (35). Whether this decrease is a direct consequence of the CFTR mutation or an artifact of an
underlying infection cannot be ascertained; however,
these observations fuel speculation that the CFTR mutation might indirectly attenuate lung antioxidant defenses. Recently, experiments using pulmonary epithelial cell lines demonstrated that cells containing
defective CFTR secreted less GSH than control cells
containing functional CFTR and that transfection of
these cells with functional CFTR restored GSH secretion to that of control cells (16). These data provide
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society
L31
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
Velsor, Leonard W., Anna van Heeckeren, and Brian
J. Day. Antioxidant imbalance in the lungs of cystic fibrosis
transmembrane conductance regulator protein mutant mice.
Am J Physiol Lung Cell Mol Physiol 281: L31–L38,
2001.—Recent studies suggest that the cystic fibrosis (CF)
transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and
when defective creates an antioxidant imbalance. To test
whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue
from CFTR knockout (CFTR-KO) and wild-type (WT) mice
were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and ␥-glutamyltransferase. In the CFTR-KO mice, the ELF concentration of
GSH was decreased (51%) compared with that in WT mice.
The concentration of GSH in the lung tissue of CFTR-KO
mice, however, was not significantly different from that in
WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were
significantly increased compared with those in WT mice (48
and 28%, respectively). Tissue lipid and DNA oxidation were
evaluated by measurement of thiobarbituric acid-reactive
substances and 8-hydroxy-2⬘-deoxyguanosine, respectively.
The levels of thiobarbituric acid-reactive substances and
8-hydroxy-2⬘-deoxyguanosine in the lung tissue of CFTR-KO
mice were significantly increased compared with those in WT
mice. These data support our hypothesis that a mutation in
the CFTR gene can affect the antioxidant defenses in the
lung and may contribute to the exaggerated inflammatory
response observed in CF.
L32
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS
METHODS
Mice. The CFTR-KO mice used in this study were congenic
B6.129P2-Cftrtm1Unc, which possess the S489X mutation in
CFTR that renders the CFTR protein nonfunctional (9, 40).
Heterozygous breeding pairs were either originally obtained
from Jackson Laboratories (Bar Harbor, ME) or a kind gift
from Sandra Gendler (Mayo Clinic Scottsdale, Scottsdale,
AZ). In this study, the CFTR-KO mice, homozygous for the
S489X mutation, were compared with their WT littermates.
Genotypes for each mouse were determined by PCR with
DNA isolated from tail clips as described in DNA extraction
and genotyping by PCR. CFTR-KO mice were maintained on
a liquid elemental diet (Peptamen, Nestle, Glendale, CA).
WT mice received the regular solid mouse chow (Teklad 9F
sterilizable rodent diet 8760, Harland, Madison, WI) or the
liquid elemental diet (14). Autoclaved tap water in bottles
with sipper tubes was provided ad libitum, and all mice were
housed on inedible corncob bedding to prevent the intestinal
obstruction associated with the CFTR mutation (14).
DNA extraction and genotyping by PCR. DNA for genotyping was isolated from tail clips. Approximately 0.5 cm of
tissue was hydrolyzed in 50 ␮l of 0.2 N NaOH and incubated
at 75°C for 15–20 min. The samples were then neutralized
with 200 ␮l of 40 mM Tris (pH 7.5), and the debris was
removed by centrifuging for 30 s.
Murine CFTR DNA from 129/Sv, C57BL/6J, A/J, BALB/cJ,
DBA2/J, and C3H/HeJ mice was sequenced with a modification of previously published methods (40). In the CFTR
sequence from 129/Sv mice, single nucleotide differences in
exons 14a and 17a alter the RsaI and AluI restriction sites,
respectively. Although both exons amplify very efficiently,
exon 14a is closer to the actual CFTR mutations. With a
single PCR assay, WT and CFTR-KO mice can be easily
distinguished by restriction of the PCR product and size
separation on agarose.
A PCR master mix [1.5 ␮l of 10⫻ PCR buffer, 0.6 ␮l of
primer, 0.2 ␮l of 5 U/ml of Taq polymerase (GIBCO BRL, Life
Technologies, Grand Island, NY), 1.2 ml of deoxynucleotide
triphosphates, 8.6 ml of water, and 0.3 ml of magnesium
chloride per 2 ␮l of tail sample] was used for genotyping. The
PCR primers for RsaI, mCFEx14a5⬘ (GAG TGT TTT CTT
GAT GAT GTG) and mCFEx14a3⬘ (ACC TCA ACC AGA AAA
ACC AG), were obtained from Integrated DNA Technologies
(Coralville, IA) at a concentration of 20 nM each. Restriction
buffer was made with 2 ␮l of 10 U/ml of RsaI and 2 ␮l of 10⫻
RsaI buffer (10 mM MgCl2, 10 mM bis-Tris propane-HCl, pH
7.0, and 1 mM dithiothreitol) in 2.8 ␮l of water per sample.
PCR products were separated on an agarose gel (1% agarose
and 2% NuSieve) in Tris-acetate-EDTA buffer. Bands at 109
and 21 bp were identified as WT, and an undigested 130-bp
band was identified as 129/Sv-derived CFTR mutants.
Isolation of BALF and lung tissue. The ELF was sampled
for antioxidant concentrations with BALF and normalized
for dilution with the urea method (8). The mice were killed by
carbon dioxide anoxia followed by exsanguination by direct
cardiac puncture. BALF was collected with three individual
1-ml aliquots of sterile phosphate-buffered saline (PBS), pH
7.4. The three aliquots were pooled, acidified with 5% metaphosphoric acid, and centrifuged (4,000 g for 5 min at 4°C) to
remove cells; the supernatant was retained and stored at
⫺80°C for subsequent analyses. The right and left lungs were
then removed and snap-frozen in liquid nitrogen. The lungs
were then ground into a fine powder with a liquid nitrogencooled mortar and pestle and then stored at ⫺70°C until
analysis. Aliquots of the ground tissue were carefully removed under liquid nitrogen as needed for subsequent analyses.
GSH assay in BALF and lung tissue. To minimize GSH
loss, BALF was acidified with 5% metaphosphoric acid (150
␮l/ml), cooled on ice, and centrifuged (10,000 g for 10 min at
4°C) to remove precipitated proteins. To determine GSH
concentrations in lung tissue, ⬃20 mg of the ground tissue
were dissolved in 600 ␮l of PBS; this solution was then
acidified with 5% metaphosphoric acid, cooled, and centrifuged to remove precipitated proteins. GSH concentrations in
the concentrated BALF and tissue homogenates were then
determined spectrophotometrically with a commercially
available assay that forms a chromogen with GSH (GSH-400,
Oxis International, Portland, OR). Determination of the
GSH-to-oxidized glutathione (GSSG) ratio in ELF was calculated from the difference in GSH concentrations in the BALF
treated with 0.5 mU/ml of GR and 10 ␮M NADPH (28).
Serum and BALF urea concentrations. To determine actual ELF concentrations of soluble antioxidants from BALF,
a dilution factor was derived from the difference between
BALF and serum urea concentrations. It is assumed that the
urea concentrations in the vascular and ELF compartments
are equivalent because urea is freely diffusible (8). A dilution
factor is thereby obtained by dividing the serum urea concentration by the BALF concentration. ELF concentrations
are then calculated by multiplying the BALF concentration
by the dilution factor. Urea concentrations in the samples
were determined with a commercially available reagent (Sigma Diagnostics, St. Louis, MO).
Lung GR activity. To determine GR activity in the lung,
10–25 mg of ground lung tissue were dissolved in 800 ␮l of
cold homogenization buffer (50 mM potassium phosphate
and 1 mM EDTA, pH 7.5) and centrifuged (8,500 g for 10 min
at 4°C), and the supernatant was retained for analysis. GR
activity in the lung homogenate was determined spectrophotometrically (340 nm) from the rate of NADPH consumption
by GR in the reduction of GSSG with a commercially available kit (Oxis International). GR activity is expressed as
units per milligram of sample protein (Coomassie Plus,
Pierce, Rockford, IL). The pellets from these homogenates
were utilized for the determination of ␥-GT activity (see Lung
␥-GT activity).
Lung GPx activity. To determine GPx activity in the lung,
10–35 mg of ground lung tissue were dissolved in 1.0 ml of
cold homogenization buffer (50 mM Tris 䡠 HCl, 5 mM EDTA,
and 1 mM 2-mercaptoethanol, pH 7.5) and centrifuged (7,500
g for 15 min at 4°C), and the supernatant was retained for
analysis. The GPx activity in the homogenate was determined with a commercially available kit (Oxis International)
to which t-butyl hydroperoxide was added as a GPx substrate
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
support for the hypothesis that the CFTR protein is
involved in antioxidant homeostasis of the ELF.
To test whether a defect in CFTR alters the constitutive lung antioxidant defenses in vivo, ELF and lung
tissue from CFTR knockout (CFTR-KO) and wild-type
(WT) mice were compared. In the ELF, the concentration of GSH was significantly decreased in the
CFTR-KO mice, whereas tissue concentrations of GSH
were similar. In the CFTR-KO lung, the activities of
glutathione reductase (GR) and glutathione peroxidase
(GPx) were increased, whereas the activity of ␥-glutamyltransferase (␥-GT) was unchanged. Two indicators of oxidative stress, thiobarbituric acid reactive
substances (TBARS) and 8-hydroxy-2-deoxyguanosine
(8-OHdG), were also increased in the CFTR-KO lung
tissue.
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS
RESULTS
GSH concentrations in the ELF and lung tissue. The
ELF GSH and GSSG concentrations in WT and
CFTR-KO mice were calculated from their respective
concentrations in BALF (Fig. 1A). Because BALF is a
manyfold dilution of the actual ELF, serum and BALF
urea concentrations were measured to determine the
ELF dilution factor. No differences in BALF or serum
urea concentrations between the WT and CFTR-KO
mice were observed (data not shown). ELF GSH concentration in the WT mice was 512 ⫾ 63 ␮M and in
agreement with previously published data (7). The
Fig. 1. Reduced (GSH) and oxidized glutathione (GSSG) concentrations (A) and GSH-to-GSSG (GSH/GSSG) ratios (B) in epithelial
lining fluid (ELF) of wild-type (WT) and cystic fibrosis transmembrane conductance regulator knockout (CFTR-KO) mice (n ⱖ 5). ELF
concentrations in WT and CFTR-KO mice were 512 ⫾ 63 and 249 ⫾
59 ␮M, respectively, for GSH and 106 ⫾ 15 and 41 ⫾ 18 ␮M,
respectively, for GSSG. Both GSH and GSSG concentrations were
significantly decreased in CFTR-KO mice (* P ⫽ 0.015 and 0.024,
respectively). GSH/GSSG ratios in WT and CFTR-KO mice (8.6 ⫾ 1.7
and 13.4 ⫾ 2.6, respectively; n ⱖ 8) were not significantly different
(P ⫽ 0.12).
CFTR-KO mice demonstrated a significant decrease
(51%) in ELF GSH, with a mean concentration of 244 ⫾
59 ␮M (Fig. 1A). There was also a significant decrease
(60%) in the ELF GSSG concentration in the CFTR-KO
mice (Fig. 1A). In contrast to the decrease in ELF GSH
concentration, the concentration of GSH in the lungs of
CFTR-KO and WT mice was not significantly different
(Fig. 2A). Although the CFTR-KO mice were maintained on a liquid diet, this diet did not adversely affect
the GSH concentration in the ELF of WT mice compared with that in WT mice maintained on the regular
solid diet (data not shown).
Activities of lung antioxidant enzymes. The activities
of three GSH-utilizing enzymes in lung tissue of WT
and CFTR-KO mice were compared. No differences in
the lung protein concentrations were observed in the
lung homogenates from WT and CFTR-KO mice (data
not shown). The activity of GR, the intracellular en-
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
to generate GSSG. The rate of NADPH consumption by GR in
the subsequent reduction of GSSG was used to calculate GPx
activity. GPx activity is expressed as units per milligram of
sample protein.
Lung ␥-GT activity. To determine lung ␥-GT activity, the
pellets obtained from the homogenates for GR analysis were
resuspended in homogenization buffer (100 mM Tris 䡠 HCl, 10
mM serine, and 0.1 mM EDTA, pH 7.6) and centrifuged
(5,500 g for 10 min at 4°C), and the supernatant was retained
for ␥-GT analysis. A 200-␮l aliquot of the supernatant was
mixed with 1.0 ml of reagent solution (3.2 mM ␥-glutamyl-3carboxy-4-nitroanilide, 110 mM glycine-glycine, and 110 mM
Tris 䡠 HCl, pH 8.3). ␥-GT activity was calculated from the rate
of 3-carboxy-4-nitroaniline production, which absorbs at 405
nm (11, 37). ␥-GT activity was normalized to the supernatant
protein concentration (Coomassie Plus, Pierce).
Lung TBARS. Oxidation of tissue lipids produces various
aldehydes that can be measured colorimetrically by their
reaction with thiobarbituric acid (29). Approximately 25 mg
of ground lung tissue were dissolved in 50 mM phosphate
buffer (pH 7.4) containing 1 mM butylated hydroxytoluene.
An aliquot of the sample was then acidified with an equal
volume of phosphoric acid. Thiobarbituric acid (0.1 M) was
added, and the mixture was heated to 90°C for 45 min. The
chromogen in the sample was extracted with n-butanol, and
the absorbance at 535 nm was measured with a plate reader
(SpectraMax 340PC, Molecular Devices, Sunnyvale, CA).
TBARS in the samples were calculated from a standard curve
and normalized for protein content.
HPLC analysis for 8-OHdG in lung DNA. DNA from
mouse lung tissue was obtained by a chloroform-isoamyl
alcohol extraction of proteinase K-digested lung homogenates (38). The purified DNA was then hydrolyzed to nucleosides with nuclease P1 and alkaline phosphatase. Samples
were analyzed for 8-OHdG and 2⬘-deoxyguanosine (2-dG) by
HPLC coupled with coulometric electrochemical and ultraviolet detection (CoulArray model 5600, ESA, Chelmford, MA),
respectively (38). Sample analysis was done with a 4.6 ⫻
150-mm, C-18 reverse-phase column (YMCbasic, YMC, Wilmington, NC) with a mobile phase of 100 mM sodium acetate
in 5% methanol at pH 5.2 (38). 2-dG was detected by ultraviolet light at 265 nm, whereas 8-OHdG was detected electrochemically with electrode potentials of 285, 365 and 435
mV. Under these conditions, 2-dG and 8-OHdG had retention
times of ⬃7.4 and 9.5 min, respectively. Nucleoside concentrations were calculated from standard curves generated
daily with freshly prepared standards.
Statistical analysis. Data are presented as means ⫾ SE.
Unless noted otherwise, each experimental group contained
five samples, with each sample measured in duplicate. Data
were subsequently analyzed for significant differences with
t-tests, with significance attained when P ⱕ 0.05.
L33
L34
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS
Fig. 2. A: concentration of GSH in the lung tissue normalized to
sample weight. Although there was a small increase in the GSH
content in CFTR-KO mouse lungs compared with WT lungs (0.31 ⫾
0.03 and 0.38 ⫾ 0.03 nmol/mg lung, respectively; n ⱖ 6), the increase
was not significant (P ⫽ 0.13). B: GSH/GSSG ratios in the WT
(22.1 ⫾ 3.9) and CFTR-KO (25.8 ⫾ 1.8) mouse lungs (n ⱖ 6) were also
not significantly different (P ⫽ 0.46).
zyme responsible for reducing GSSG to GSH, was significantly increased in CFTR-KO mouse lungs (Fig. 3).
GR activity increased from 2.5 ⫾ 0.2 U/mg protein in
WT mice to 3.8 ⫾ 0.3 U/mg protein in CFTR-KO mice.
GPx, the intracellular enzyme that utilizes GSH to
reduce peroxides, was also significantly elevated
CFTR-KO mouse lungs. GPx activity increased from
338 ⫾ 20 U/mg protein in WT mouse lungs to 431 ⫾ 28
U/mg protein in CFTR-KO mouse lungs (Fig. 4). The
process of extracellular catabolism of GSH for subsequent cellular processes, including GSH synthesis, is
mediated by ␥-GT, which cleaves GSH to yield the
␥-Glu and Cys-Gly moieties (33). In the CFTR-KO
mouse lung, ␥-GT was not significantly altered over
that in WT lungs (Fig. 5).
Markers of oxidative stress in the ELF. To determine
whether there was oxidative stress in the ELF compartment, TBARS and GSH-to-GSSG ratios in the
BALF were determined. Not surprisingly, TBARS were
below the detection limits of the spectrophotometric
assay in both the WT and CFTR-KO mice (data not
shown). A change in the GSH-to-GSSG ratio can often
be an indicator of oxidative stress (2, 28). Because the
decreases in GSH and GSSG were relatively proportional between the WT and CFTR-KO mice (Fig. 1A),
the GSH-to-GSSG ratios did not differ significantly
and suggested the absence of oxidative stress in the
ELF of CFTR-KO mice (Fig. 1B).
Markers of oxidative stress in lung tissue. Increases
in antioxidants and antioxidant enzymes, such as
those shown in Markers of oxidative stress in the ELF,
often occur in response to an oxidative stress (30, 32,
33, 36, 41). Consequently, mouse lung tissues from
CFTR-KO mice were analyzed for more direct indications of oxidative stress. Tissue GSH-to-GSSG ratios
were not significantly different between the mice (Fig.
2B). More direct indicators of oxidative injury, namely
TBARS and 8-OHdG, were significantly elevated in
CFTR-KO mouse lungs. The concentration of TBARS, a
Fig. 4. GSH peroxidase (GPx) activity in the lung tissue. Activity of
GPx in lung tissue was normalized to sample protein concentrations.
CFTR-KO mice had significantly more GPx activity in the lung tissue
than WT mice (431 ⫾ 28 and 338 ⫾ 20 U/mg protein, respectively;
* P ⫽ 0.012; n ⱖ 10). No differences in lung protein concentrations
were observed in these samples (data not shown).
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
Fig. 3. GSH reductase (GR) activity in the lung tissue. Activity of GR
in lung tissue was normalized to sample protein concentrations.
CFTR-KO mice had significantly greater lung GR activity than WT
mice (3.76 ⫾ 0.27 and 2.54 ⫾ 0.19 U/mg protein, respectively; * P ⫽
0.001; n ⱖ 12). No differences in lung protein concentrations were
observed in these samples (data not shown).
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS
Taken together, these two studies provide substantial
support of the hypothesis that CFTR directly regulates
the concentration of ELF GSH and may subsequently
render the CF lung more susceptible to oxidative injury.
Based on this potential link between CFTR and the
transport of a critical antioxidant into the ELF compartment, we used CFTR-KO mice to test the hypothesis that the CFTR mutation alters antioxidant defenses in the lung. The CFTR-KO mouse used in this
study (B6.129P2-Cftrtm1Unc) possesses a mutation that
renders the CFTR protein nonfunctional (9, 40, 43, 44).
The decreased concentration of GSH in the ELF of
CFTR-KO mice indicates that this defect in CFTR
alters GSH availability in the ELF. Thus the previously reported decrements in the concentration of GSH
in BALF may not only be due to the infection-induced
oxidative stress but may also be a direct result of
marker for lipid oxidation, was increased over 25% in
the lung tissue of the CFTR-KO mice (Fig. 6A). Levels
of 8-OHdG, formed from the oxidation of 2-dG in DNA,
were also significantly increased in CFTR-KO mouse
lungs (Fig. 6B).
DISCUSSION
This study demonstrates that a mutation in the
CFTR gene decreases the concentration of GSH in the
ELF, increases the activities of lung antioxidant enzymes, and possibly stimulates oxidative stress.
CFTR-KO mice demonstrated a significant decrease in
ELF GSH compared with WT mice. Although the lung
tissue did not exhibit any alterations in GSH concentration, the CFTR-KO mice exhibited significant increases in GR and GPx activities. Elevated activities of
such antioxidant enzymes are often an adaptive response to oxidative stress (30, 32, 33, 36, 41). Increased
oxidation of lipids and DNA in the lungs of CFTR-KO
mice also suggests the presence of an oxidative stress.
Although studies (18, 34, 35) have documented that
ELF concentrations of GSH are considerably lower in
normal than in adult CF patients, a direct link to
CFTR has been difficult to establish. Even in the absence of overt clinical indications of an infection, many
CF patients exhibit evidence of an underlying inflammatory process occurring in their lungs (1, 19). This
underlying inflammation has limited the use of these
data to support a hypothesis that CFTR was directly
involved in the transport or regulation of GSH in the
ELF. However, two recent in vitro studies (16, 25) have
provided strong evidence that CFTR is associated with
the transport of GSH. In the first study, Linsdell and
Hanrahan (25) demonstrated that in addition to Cl⫺
and other anions, GSH could permeate the cell membrane through CFTR in vitro. The subsequent in vitro
study (16) used airway cells isolated from a CF patient
and demonstrated that cells lacking functional CFTR
had significantly less GSH in their apical fluid than
normal cells or cells transfected with functional CFTR.
Fig. 6. Concentration of thiobarbituric acid-reactive substances
(TBARS; A) and 8-hydroxy-2⬘-deoxyguanosine (8-OHdG; B) in lung
tissue of CFTR-KO and WT mice. A: level of TBARS was significantly
elevated in the lung tissue of CFTR-KO mice compared with WT
mice (100.0 ⫾ 4.0 and 126.6 ⫾ 8.0%, respectively; * P ⫽ 0.0251). B:
ratio of 8-OHdG to every 105 2⬘-deoxyguanosine (2-dG) in DNA
isolated from lung tissue. Levels of 8-OHdG were significantly increased in DNA from CFTR-KO lungs compared with those in WT
lungs (5.67 ⫾ 0.94 and 3.72 ⫾ 0.37 8-OHdG per 105 2-dG, respectively; * P ⫽ 0.0459).
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
Fig. 5. ␥-Glutamyltransferase (␥-GT) activity in lung tissue. Activity
of ␥-GT in lung tissue was normalized to sample protein concentration. Lung ␥-GT activity in the CFTR-KO mice was not significantly
increased compared with that in WT mice (P ⫽ 0.159). No differences
in lung protein concentrations were observed in these samples (data
not shown).
L35
L36
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS
decreased. The decrease, however, was nearly proportional to the decrease in GSH and did not significantly
alter the GSH-to-GSSG ratio from that of the WT mice.
Calculating a GSH-to-GSSG ratio from ELF GSH and
GSSG concentrations reported in a previous study with
rats (5) produces a ratio of 8.6; this is exactly the same
value reported here for the WT mice. Therefore, the
decreased ELF concentration of GSH in the CFTR-KO
mice is not likely due to increased oxidation but rather
is a direct result of impaired epithelial transport.
In contrast to the decreased antioxidant capacity of
the ELF, the lung tissue demonstrated increases in the
antioxidant enzymes GR and GPx. An increase in these
enzymes usually occurs as an adaptive response to
increased oxidative stress. GR functions to recycle
GSH by using NADPH to reduce GSSG back to GSH.
Increases in GR activity have been demonstrated under conditions of oxidative stress in many tissues including the lung (24). Under oxidative stress, upregulation of GR can maintain GSH-to-GSSG ratios as an
adaptive response. GPx is another enzyme upregulated
by oxidative stress that functions to eliminate oxidizing peroxides, including H2O2 and lipid peroxides (3,
24). Together, these enzymes can neutralize injurious
oxidants with GSH and replenish GSH through increased GSSG reduction. The upregulation of this enzyme system is indicative of the presence of an increased oxidant burden in the CFTR-KO lung.
␥-GT is an enzyme found on the luminal surfaces of
Clara cells and, to a lesser extent, type II cells of the
alveolar epithelium (12). ␥-GT in conjunction with a
dipeptidase serves to recycle GSH by cleaving it into its
three amino acids that are readily transported to the
intracellular compartment (17). Oxidative stress produces an increase in ␥-GT activity in both in vitro and
in vivo models (17, 22). Consequently, increases in
␥-GT activity are regarded as an indication of oxidative
stress. Although the increase in lung ␥-GT activity
observed in CFTR-KO mice was not significant, the
increased activity of ␥-GT is not inconsistent with the
GR and GPx data that suggest the presence of an
oxidative stress.
More direct markers of oxidative injury provide evidence of an inherent oxidative stress in the CFTR-KO
mice. In this study, TBARS and 8-OHdG were utilized
as markers of oxidative stress in lung tissue. TBARS in
CFTR-KO mouse lung tissue were significantly increased compared with those in WT mouse lungs. This
result is consistent with a previous study (31) that
found increases in plasma TBARS in children with CF.
It could be argued, however, that the elevated TBARS
levels could be a direct result of ongoing infections or
dietary effects. Another study with CF patients without clinical signs of exacerbations, however, demonstrated increases in plasma TBARS, hydroperoxides,
and protein carbonyls and erythrocyte GR and superoxide dismutase activities (13). These studies clearly
indicate the existence of an oxidative stress in CF and
provide support for the increased TBARS observed
here. Although the previous studies indicate the presence of an oxidative stress, the fact that the results
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
impaired GSH transport into the ELF compartment
(25, 35).
The pancreatic dysfunction associated with CF leads
to inadequate breakdown and absorption of the fatsoluble nutrients such as vitamin E, ␤-carotene, and
selenium and may contribute to the antioxidant imbalances observed in CF (30). In addition to the pulmonary manifestations, CF patients also suffer from gastrointestinal obstructions and malabsorption of
nutrients (10, 42). To minimize these problems in the
CFTR-KO mice, the mice were housed on inedible
bedding and maintained on a liquid diet (14). WT mice
maintained on either the regular solid diet or the liquid
diet did not exhibit significant differences in ELF GSH
concentration. In addition, CFTR-KO mice with gutcorrected CFTR (46) also have a decreased concentration of GSH in their ELF (data not shown). Because
there is little effect of diet in WT mice, the observed
difference between the WT and CFTR-KO mice is most
likely due to the lack of functional CFTR protein in the
knockout mice and not attributed to the liquid diet on
which the mice were maintained.
As a consequence of the reduced ELF concentration
of GSH, the CF lung may be more vulnerable to oxidative stress that results from an infectious agent (35).
After invasion of an infectious agent, there is an influx
of neutrophils that generate bactericidal oxidants such
as superoxide, hydrogen peroxide, and hypochlorous
acid. Extracellular antioxidants such as GSH can protect the underlying epithelial cells from these oxidative
processes (34, 35, 42). Factors contributing to an imbalance between oxidant production and antioxidant
defense yield a net oxidative stress, which can cause
epithelial injury concomitant to the bacterial killing.
Due to epithelial damage and release of intracellular
contents, the inflammatory response could be further
stimulated and exacerbate the condition (30). Compared with WT mice, CFTR-KO mice infected with
Pseudomonas have higher mortality rates and markedly elevated levels of inflammatory mediators (44). In
CF patients, lung infections elicit an intense inflammatory response characterized by an influx of neutrophils and the secretion of cytokines (21). Clearing a
pulmonary infection is further complicated in CF patients by the thickened characteristics of the ELF mucus (39). GSH is the predominant mucolytic agent in
the ELF; its deficiency, due to lack of functional CFTR,
probably contributes to thickened ELF mucus in CF
patients. The continued presence of the bacteria to
drive inflammation coupled with decreased protection
by antioxidants favors destruction of tissue. Thus the
inherent GSH deficiency caused by impaired epithelial
transport coupled with insufficient dietary absorption
of vital antioxidants may seriously predispose the CF
lung to oxidative injury (34, 35, 42).
The lower concentration of GSH in the ELF of
CFTR-KO mice is not due, however, to an inherent
oxidative stress present in the ELF. A change in the
ratio of GSH to GSSG is often used as an indicator of
oxidative stress (2, 28). In this study, the GSSG concentration in CFTR-KO mouse lungs was significantly
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS
onstrated in this study suggests that an oxidative
stress is occurring within the cell, it is unclear whether
the oxidized DNA is nuclear, mitochondrial, or both.
Studies currently underway in our laboratory will delineate the source of the 8-OHdG.
The exact mechanism by which CFTR may mediate
oxidative stress in the mitochondria is unclear. Because mitochondria lack GSH-synthesizing enzymes,
all intramitochondrial GSH must be transported from
the cytosol. Currently, the mechanism by which GSH is
transported into the mitochondria has not been identified. Consequently, it is possible that in addition to
apical transport of GSH, the CFTR protein may also
facilitate GSH transport into the mitochondria directly
or through the modulation of proteins involved in its
transport. If this is true, then an imbalance in mitochondrial GSH may be present and provide a link
between the CFTR protein and mitochondrial oxidative
stress.
The decreased concentration of ELF GSH observed
in CF patients has led some investigators to propose
that restoring GSH concentrations in this compartment may reduce tissue destruction (34). In this study,
application of aerosolized GSH increased the BALF
concentration of GSH and reduced phorbol 12-myristate 13-acetate-stimulated superoxide release in macrophages from CF patients. In addition to reducing
oxidant-mediated epithelial injury, increased ELF
GSH concentrations may also limit tissue destruction
by preserving the antiproteases that are present in the
ELF (4, 21). Given the likelihood of developing severe
pulmonary infections, augmentation of the ELF GSH
pool is likely to be critical to improving the lifespan of
CF patients. The results of this study demonstrate, in
an animal model defective in the CFTR protein, decreased GSH concentrations in the ELF and increased
oxidation of lung lipids and DNA. These data support
the hypothesis that CFTR regulates lung antioxidants
and, when defective, may contribute to oxidative stress
associated with CF.
We thank Merle Fleischer, Mark Goldstein, Lisa Hogue, Todd
Romigh, and Christiaan van Heeckeren for expert technical assistance in these studies and Drs. Frank Accurso and Pamela B. Davis
for helpful suggestions. We also thank Tanya Canafax for secretarial
support.
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-59602 and HL-31992 (to B. J. Day);
National Institute of Diabetes and Digestive and Kidney Diseases
Grant DK-27651 (to A. V. Heeckeren); and research and development grants from the Cystic Fibrosis Foundation (to A. V. Heeckeren
and B. J. Day).
REFERENCES
1. Armstrong DS, Grimwood K, Carzino R, Carlin JB, Olinsky A, and Phelan PD. Lower respiratory infection and inflammation in infants with newly diagnosed cystic fibrosis. BMJ 310:
1571–1572, 1995.
2. Arseni M, Sastre J, Pallardo FV, Lloret A, Lehner M,
Asuncion G, and Vina J. Ratio of reduced to oxidized glutathione as indicator of oxidative status and DNA damage. Methods
Enzymol 299: 267–276, 1999.
3. Avissar N, Finkelstein JN, Horowitz S, Willey JC, Coy E,
Frampton MW, Watkins RH, Khullar P, Xu Y, and Cohen
HJ. Extracellular glutathione peroxidase in human lung epithe-
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
were obtained from plasma limits identification of the
site of the oxidative events. The results reported here,
however, extend these previous observations and identify the lung as a potential site of oxidative events. A
more sensitive indicator of oxidative stress is the detection of oxidized bases, particularly 8-OHdG, in DNA
(15, 38). A previous study (6) demonstrating elevated
levels of 8-OHdG in the urine of CF patients also
suggests the presence of an oxidative stress. Moreover,
no correlation between clinical indicators of lung function and increases in 8-OHdG was identified, which
suggests that CF patients have an inherent oxidative
stress independent of clinical causes such as infection.
In our study, a small but significant increase in
8-OHdG was detected in DNA from CFTR-KO mouse
lungs. These data suggest that a mild oxidative stress
is occurring in the lung tissue and provide a plausible
explanation for the elevation in lung GR and GPx in
CFTR-KO mice.
Although the elevated levels of TBARS and 8-OHdG
demonstrate the presence of an oxidative stress in the
lung tissue of CFTR-KO mice, the GSH-to-GSSG ratio
remains unchanged. The GSH-to-GSSG ratio in the
CFTR-KO lungs was not significantly different from
WT lungs and was only slightly greater than the ratios
recently reported (GSH/GSSG ⬇ 19) for rat lungs (23).
The lack of any change in this parameter suggests the
absence of oxidative stress and is in contradiction to
the other markers. The most plausible explanation for
this divergence is that the increased GR activity compensated for any elevations in GSSG formation and
thereby maintained normal GSH-to-GSSG ratios. Thus
the oxidative stress indicated by TBARS and 8-OHdG
may alter the GSH-to-GSSG ratio by increasing GSSG
production, but the increased GR activity negates its
detection.
The previous studies demonstrating the presence of
an oxidative stress in the absence of clinical indicators
of infection suggest that oxidative stress may be inherent to CF. The data presented here also demonstrate
the presence of an oxidative stress; however, effects
from infection and diet are largely minimized and
provide more convincing evidence that a mild inherent
lung oxidative stress occurs in CF. This mild oxidative
stress itself produces little lung pathology but may
sensitize the lung to overtly respond to stimuli such as
infections. Although the data presented here provide
evidence of oxidative stress in the lung, they do not
identify the source of oxidative stress. It is well known
that mitochondrial electron transport can be a significant source of cellular oxidants such as superoxide and
hydrogen peroxide under both normal and pathological
conditions. CF patients demonstrated increased oxygen consumption and increased energy requirements
consistent with mitochondrial dysfunction (45). In that
study, cells isolated from CF patients demonstrate
increased oxygen consumption and activity of NADH
oxidase and NADH dehydrogenase in mitochondria.
These data provide further evidence that events in the
mitochondria may contribute to injurious oxidative
events. Although the increased oxidation of 2-dG dem-
L37
L38
4.
5.
6.
7.
8.
9.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
lial lining fluid and in lung cells. Am J Physiol Lung Cell Mol
Physiol 270: L173–L182, 1996.
Birrer P. Proteases and antiproteases in cystic fibrosis: pathogenetic considerations and therapeutic strategies. Respiration
62: 25–28, 1995.
Boehme DS, Hotchkiss JA, and Henderson RF. Glutathione
and GSH-dependent enzymes in bronchoalveolar lavage fluid
cells in response to ozone. Exp Mol Pathol 56: 37–48, 1992.
Brown RK, McBurney A, and Kelly FJ. Oxidative damage to
DNA in patients with cystic fibrosis. Free Radic Biol Med 18:
801–806, 1995.
Cantin AM, North SL, Hubbard RC, and Crystal RG. Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol 63: 152–157, 1987.
Chinard FP. Quantitative assessment of epithelial lining fluid
in the lung. Am J Physiol Lung Cell Mol Physiol 263: L617–
L618, 1992.
Clarke LL, Grubb BR, Gabriel SE, Smithies O, Koller BH,
and Boucher RC. Defective epithelial chloride transport in a
gene targeted mouse model of cystic fibrosis. Science 257: 1125–
1128, 1992.
Davis PB, Drumm M, and Konstan MW. Cystic fibrosis. Am J
Respir Crit Care Med 154: 1229–1256, 1996.
Day BJ and Carlson GP. Kinetic comparison of rat pulmonary
and renal gamma-glutamyl transpeptidase activities. Res Commun Chem Pathol Pharmacol 70: 371–374, 1990.
Dinsdale D, Green JA, Manson MM, and Lee MJ. The ultrastructural immunolocalization of gamma-glutamyltranspeptidase
in rat lung: correlation with the histochemical demonstration of
enzyme activity. Histochem J 24: 144–152, 1992.
Dominguez C, Gartner S, Linan S, Cobos N, and Moreno A.
Enhanced oxidative damage in cystic fibrosis patients. Biofactors
8: 149–153, 1998.
Eckman E, Cotton C, Kube D, and Davis PB. Dietary
changes improve survival of CFTR S489X homozygous mutant
mouse. Am J Physiol Lung Cell Mol Physiol 269: L625–L630,
1995.
Floyd RA, West MS, Eneff KL, Schneider JE, Wong PK,
Tingey DT, and Hogsett WE. Conditions influencing yield and
analysis of 8-hydroxy-2⬘-deoxyguanosine in oxidatively damaged
DNA. Anal Biochem 188: 155–158, 1990.
Gao L, Kim KJ, Yankaskas JB, and Forman HJ. Abnormal
glutathione transport in cystic fibrosis airway epithelia. Am J
Physiol Lung Cell Mol Physiol 277: L113–L118, 1999.
Griffith OW. Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic Biol Med 27: 922–935,
1999.
Hull J, Vervaart P, Grimwood K, and Phelan P. Pulmonary
oxidative stress response in young children with cystic fibrosis.
Thorax 52: 557–560, 1997.
Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, and
Riches DW. Early pulmonary inflammation in infants with
cystic fibrosis. Am J Respir Crit Care Med 151: 1075–1082, 1995.
Konstan MW and Berger M. Current understanding of the
inflammatory process in cystic fibrosis: onset and etiology. Pediatr Pulmonol 24: 137–161, 1997.
Konstan MW, Hilliard KA, Norvell TM, and Berger M.
Bronchoalveolar lavage findings in cystic fibrosis patients with
stable, clinically mild lung disease suggest ongoing infection and
inflammation. Am J Respir Crit Care Med 150: 448–454, 1994.
Kugelman A, Choy HA, Liu R, Shi MM, Gozal E, and
Forman HJ. Gamma glutamyl transpeptidase is increased by
oxidative stress in rat alveolar L2 epithelial cells. Am J Respir
Cell Mol Biol 11: 586–592, 1994.
Lakritz J, Plopper CG, and Buckpitt AR. Validated highperformance liquid chromatography-electrochemical method for
determination of glutathione and glutathione disulfide in small
tissue samples. Anal Biochem 247: 63–68, 1997.
Lee J, Mustafa MG, and Afifi AA. Effects of short-term, single
and combined exposure to low-level NO2 and O3 on lung tissue
enzyme activities in rats. J Toxicol Environ Health 29: 293–305,
1990.
Linsdell P and Hanrahan JW. Glutathione permeability of
CFTR. Am J Physiol Cell Physiol 275: C323–C326, 1998.
26. Ma J and Davis PB. What we know and what we do not know
about cystic fibrosis transmembrane conductance regulator. Clin
Chest Med 19: 459–469, 1998.
27. Mickle JE and Cutting GR. Clinical implications of cystic
fibrosis transmembrane conductance regulator mutations. Clin
Chest Med 19: 443–455, 1998.
28. O’Donovan DJ, Katkin JP, Tamura T, Husser R, Xu X,
Smith CV, and Welty SE. Gene transfer of mitochondrially
targeted glutathione reductase protein H441 cells from t-butyl
hydroperoxide-induced oxidant stresses. Am J Respir Cell Mol
Biol 20: 256–263, 1999.
29. Ohkawa H, Ohishi N, and Yagi K. Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Anal Biochem 95:
351–358, 1979.
30. Poli G and Parola M. Oxidative damage and fibrogenesis. Free
Radic Biol Med 22: 287–305, 1997.
31. Portal BC, Richard MJ, Faure HS, Hadjian AJ, and Favier
AE. Altered antioxidant status and increased lipid peroxidation
in children with cystic fibrosis. Am J Clin Nutr 61: 843–847,
1995.
32. Quinlan T, Spivack S, and Mossman BT. Regulation of antioxidant enzymes in lung after oxidant injury. Environ Health
Perspect 102: 79–87, 1994.
33. Rahman I and MacNee W. Lung glutathione and oxidative
stress: implications in cigarette smoke-induced airway disease.
Am J Physiol Lung Cell Mol Physiol 277: L1067–L1088, 1999.
34. Roum JH, Borok Z, McElvaney NG, Grimes GJ, Bokser
AD, Buhl R, and Crystal RG. Glutathione aerosol suppresses
lung surface inflammatory cell-derived oxidants in cystic fibrosis. J Appl Physiol 87: 438–443, 1999.
35. Roum JH, Buhl R, McElvaney NG, Borok Z, and Crystal
RG. Systemic deficiency of glutathione in cystic fibrosis. J Appl
Physiol 75: 2419–2424, 1993.
36. Sagara Y, Dargusch R, Chambers D, Davis J, Schubert D,
and Maher P. Cellular mechanisms of resistance to chronic
oxidative stress. Free Radic Biol Med 24: 1375–1389, 1998.
37. Shaw L. ␥-Glutamyltransferase. In: Methods of Enzymatic
Analysis, edited by Bergmeyer HU, Bermeyer J, and Grassl M.
Deerfield Beach, FL: Verlag-Chemie, 1983, p. 349–364.
38. Shigenaga MK, Aboujaoude EN, Chen Q, and Ames BN.
Assays of oxidative DNA damage biomarkers 8-oxo-2⬘-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological
fluids by high-performance liquid chromatography with electrochemical detection. Methods Enzymol 234: 16–33, 1994.
39. Smith JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic
fibrosis airway epithelia fail to kill bacteria because of abnormal
airway surface fluid. Cell 82: 229–236, 1996.
40. Snouwaert JN, Brigman KK, Latour AM, Malouf NN,
Boucher RC, Smithies O, and Koller BH. An animal model
for cystic fibrosis made by gene targeting. Science 257: 1083–
1088, 1992.
41. Tian L, Cai Q, and Wei J. Alterations of antioxidant enzymes
and oxidative damage to macromolecules in different organs of
rats following aging. Free Radic Biol Med 24: 1477–1484, 1998.
42. Van der Vliet A, Eiserich JP, Marelich GP, Halliwell B,
and Cross CE. Oxidative stress in cystic fibrosis: does it occur
and does it matter? Adv Pharmacol 38: 491–513, 1997.
43. Van Heeckeren AM, Tscheikuna J, Walenga RW, Konstan
MW, Davis PB, Erokwu B, Haxhiu MA, and Ferkol TW. Effect
of Pseudomonas infection on weight loss, lung mechanics, and
cytokines in mice. Am J Respir Crit Care Med 161: 271–279, 2000.
44. Van Heeckeren AM, Walenga RW, Konstan MW, Bonfield
T, Davis PB, and Ferkol TW. Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with
Pseudomonas aeruginosa. J Clin Invest 100: 2810–2815, 1997.
45. Von Ruecker AA, Bertele R, and Harms K. Calcium metabolism and cystic fibrosis: mitochondrial abnormalities suggest a
modification of the mitochondrial membrane. Pediatr Res 18:
594–599, 1984.
46. Zhou L, Dey C, Wert S, DaVall M, Frizzell RA, and Whitsett
J. Correction of lethal intestinal defect in mouse model of cystic
fibrosis by human CFTR. Science 266: 1705–1708, 1994.
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 14, 2017
10.
ANTIOXIDANT IMBALANCE IN CFTR KNOCKOUT MOUSE LUNGS