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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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NRF2 is a transcription factor important in the protection
against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes.
This study was designed to determine the role of NRF2 in the
pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95-98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2
/) and wild-type mice (Nrf2+/+). Pulmonary hyperpermeability, macrophage
inflammation, and epithelial injury in Nrf2/ mice were 7.6-fold,
47%, and 43% greater, respectively, compared with Nrf2+/+
mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of
NRF2 in the lungs of Nrf2+/+ mice. mRNA expression for ARE-
responsive lung antioxidant and phase 2 enzymes was evaluated
in both genotypes of mice to identify potential downstream
molecular mechanisms of NRF2 in hyperoxic lung responses.
Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2/ mice compared with Nrf2+/+ mice. Consistent with differential mRNA expression, NQO1 and total GST activities were
significantly lower in Nrf2/ mice compared with Nrf2+/+ mice
after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in
mice, possibly through transcriptional activation of lung antioxidant defense enzymes.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reactive oxygen species (ROS) have been implicated in
the pathogenesis of many acute and chronic pulmonary
disorders such as adult respiratory distress syndrome and
bronchopulmonary dysplasia (1). In laboratory animals,
administration of pure oxygen (> 95%, hyperoxia) causes
extensive pulmonary damage characterized by inflammation and death of capillary endothelial and alveolar epithelial
cells resulting in pulmonary edema and severe impairment of respiratory functions (2, 3). Sufficiently long exposure (
3 d) to hyperoxia is lethal to animals (4). The precise molecular mechanism(s) by which hyperoxia produces
lung injury remain(s) unresolved. However, excess production of ROS that could overwhelm endogenous pulmonary
antioxidant defense systems has been proposed (5), and a
number of studies have focused on enzymatic defense
components in the pathogenesis of oxygen-induced lung
damage (6).
In laboratory rodents, hyperoxia causes increases of "classic" antioxidant enzymes (e.g., superoxide dismutase [SOD], glutathione peroxidase [GPx], glutathione reductase [GR], and catalase) in the lung (6, 7). The protective roles of these enzymes in the development of oxidative lung damage have been proposed in a few in vivo studies with genetically engineered mice (i.e., gene knockout mice and transgenic mice). For example, lung inflammation and damage was attenuated in mice that overexpressed SOD3, relative to wild-type (wt) mice (8); partial protection against hyperoxic lung injury was also observed in transgenic mice overexpressing SOD2 (9). Tsan and colleagues suggested that SOD2 gene-knockout mice were more susceptible to pulmonary hyperoxic injury than normal mice (10). Enhanced pulmonary antioxidant enzyme activity through exogenous administration of SOD1 and/or catalase also provided protection to rats against hyperoxic insults (11). Heme oxygenase-1 (HO-1), an oxidative stress protein, has been also shown to be protective in hyperoxic pulmonary injury (12, 13). In addition, phase 2 detoxifying enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST) have attracted attention due to their protective roles against oxidative processes in malignant cells or tissues (14, 15). As indirect antioxidants, phase 2 enzymes detoxify reactive electrophilic metabolites, such as organic peroxides, lipid peroxides, epoxides, and quinones, and facilitate their excretion through conjugation reaction or two-electron reduction. However, little is known about the contribution of phase 2 detoxifying enzymes to lung defense against oxygen toxicity.
NF-E2-related factor 2 (NRF2) is a recently identified
cap'n'collar basic leucine zipper transcription factor. It
was originally detected in erythroid cells, but abundant
NRF2 mRNA expression has subsequently been described
in murine liver, intestine, lung, and kidney, where detoxification reactions occur routinely (16, 17). High similarity
exists between the NRF2 binding sequence (NF-E2 consensus sequence) and antioxidant response element (ARE, also referred to as electophilic response element). Consequently, NRF2 has induced mRNA expression for ARE-bearing phase 2 detoxifying enzymes such as NQO1, GST-
Ya subunit, and 
-glutamate cysteine ligase regulatory
subunit (GCLS), classic antioxidant enzymes (e.g., catalase, SOD1), and HO-1, and protected cells against carcinogenesis and oxidative stress in various in vivo (17) and in vitro (20) models. The role of NRF2 in the
pathogenesis of oxygen toxicity, however, has not been
studied in the lungs of laboratory animals.
The present study was designed to test the hypothesis
that NRF2 contributes to pulmonary protection against
hyperoxic injury in mice. Mice with site-directed mutation
(knockout) of Nrf2 (Nrf2/) and wt mice (Nrf2+/+) were
exposed to hyperoxia, and pulmonary permeability, inflammatory, and epithelial injury responses in bronchoalveolar lavage fluid (BALF) were compared in both genotypes of mice. The effects of hyperoxia exposure on lung
NRF2 mRNA expression and DNA-binding activity in wt
mice were determined by Northern blot analysis and electrophoretic mobility shift analysis (EMSA), respectively. Lung mRNA expression for selected ARE-responsive defense enzymes were examined in Nrf2/ and wt mice to
identify molecular mechanisms through which NRF2 may
contribute to the protection against oxidative lung injury.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals
Breeding pairs of ICR/Sv129-Nrf2+/ mice were obtained from a
colony at Tsukuba University (17) and maintained in the Johns Hopkins facility. Mice were fed a purified AIN-76A diet. Water was provided ad libitum. Mice were bred, and progeny were genotyped for Nrf2+/+ and Nrf2/ (17). Cages were placed in laminar flow hoods with high-efficiency particulate-filtered air. Sentinel animals were examined periodically (titers and necropsy) for
infection. All experimental protocols conducted in the mice were
performed in accordance with the standards established by the
US Animal Welfare Acts, set forth in NIH guidelines and the Policy and Procedures Manual (Johns Hopkins University School of
Hygiene and Public Health Animal Care and Use Committee).
Oxygen Exposure
Mice were placed on a fine mesh wire flooring in a sealed 45-liter
glass exposure chamber. The chamber bottom was lined with CO2 absorbent (Soda-sorb; WR Grace, Lexington, MA). Food
and water were provided ad libitum. Sufficient humidified pure
oxygen was delivered to the chamber to provide 10 changes/h
(7 liters/min flow rate). The concentration of oxygen in the exhaust
from the chamber was monitored (OM-11; Beckman, Irvine, CA)
throughout the experiments. The oxygen concentration for all experiments ranged from 95-99%. The chambers were opened
once a day for 10 min to replace CO2 absorbent, food, and water.
Age- and gender-matched (6- to 8-wk) mice of each genotype
(Nrf2+/+ and Nrf2/) were exposed to either room air or hyperoxia for 48 and 72 h (n = 4 per group).
BALF and Phenotyping
Immediately following exposure, mice were removed from the chamber, anesthetized with sodium pentobarbital (104 mg/kg), and weighed. Hyperoxia-induced changes in lungs were assessed by total protein concentration and total and differential cell counts in BALF following procedures described previously (24). Briefly, the right lung of each mouse was lavaged in situ four times with Hanks' balanced salt solution (HBSS, 17.5 ml/kg, pH 7.2- 7.4), and the recovered BALF was immediately cooled to 4°C. For each mouse, the four BALF returns were centrifuged (500 × g at 4°C), and the supernatant from the first BALF return was decanted for determination of total protein (an indicator of lung permeability). Protein concentration was measured following the method of Bradford as indicated in the manufacturer's procedure (Bio-Rad, Hercules, CA). The cell pellets from all lavage returns were combined and resuspended in 1 ml of HBSS. The numbers of cells (per ml total BALF return) were counted with a hemocytometer as indicators of lung injury and inflammation. An aliquot (200 µl) of BALF cell suspension was cytocentrifuged (Shandon Southern Products, Pittsburgh, PA) and stained with Wright-Giemsa stain (Diff-Quik; Baxter Scientific Products, McGaw Park, IL) for differential cell analysis. Differential counts for epithelial cells, macrophages, and PMNs were done by identifying 300 cells according to standard cytologic techniques (25). Epithelial cells in particular were identified by the presence of cilia.
Total Lung RNA Isolation and Northern Blot Analysis for NRF2 mRNA Expression
Total RNA was isolated from nonlavaged lung homogenate of
each mouse according to the method of Chomczynski and Sacchi
(26) as indicated in the Trizol (Life Technologies, Gaithersburg,
MD) reagent specifications. Pooled total RNA from each group
(15 µg) was separated on a 1.2% formaldehyde-agarose gel in 1×
MOPS acid buffer and transferred overnight to a nylon membrane (Nytran; Schleicher and Schuell, Keene, NH). The membrane was UV-crosslinked, and the blot was hybridized with a
double-stranded [32P]dCTP-labeled NRF2 cDNA probe (1.8 kb)
in the Perfect Hybridization Buffer (Sigma, St. Louis, MO) and
evaluated autoradiographically. As a control for loading of total
RNA, 18S RNA on the gel was examined by staining with ethidium bromide. The intensity of each NRF2 band was quantitated
using a Gel Doc 2000 System (Bio-Rad) and normalized by the
intensity of the corresponding 18S RNA band.
Lung Nuclear Protein Extraction and EMSA for NRF2 Activity
Nuclear protein extracts were prepared from pooled lung tissues
of four mice in each group as previously described (27). An aliquot of 2 µg nuclear proteins was incubated on ice with a binding
buffer (10 mM HEPES [pH 7.9], 60 mM KCl, 0.5 mM EDTA, 4%
Ficoll, 1 mM DTT, 0.2 µg PolydI-dC, 1 mM PMSF) in a total volume of 19 µl. After 15 min incubation, 1 µl (2 × 104 cpm) of
[32P]dATP end-labeled double-stranded oligonucleotide containing a NF-E2-consensus sequence (5'-TGG GGA ACC TGT
GCT GAG TCA CTG GAG-3') or ARE-consensus sequence
(5'-AGT CAC AGT GAC TCA GCA GAA TCT-3') was added
to the reaction and followed by an additional 30-min incubation
at room temperature. The mixture was subjected to electrophoresis on a 4% polyacrylamide gel with 0.25× TBE buffer for
2 h at 180 V. The gel was autoradiographed using an intensifying screen at 
70°C. The intensity of each shifted band was quantitated using a Gel Doc 2000 System (Bio-Rad).
RT-PCR for Lung Antioxidant Enzyme mRNA Expression
Total RNA (500 ng) was reverse transcribed into cDNA in a volume of 50 µl, containing 1× PCR buffer (50 mM KCl and 10 mM
Tris [pH 8.3]), 5 mM MgCl2, 1 mM each dNTPs, 125 ng oligo
(dT)15, and 50 U of Moloney Murine Leukemia Virus reverse
transcriptase (Life Technologies), at 45°C for 15 min and 95°C for
5 min using gene amp PCR System 9700 (Perkin Elmer Applied
Biosystems, Foster City, CA). Separate but simultaneous PCR
amplifications were performed with aliquots of cDNA (10 µl) at
a final concentration of 1× PCR buffer, 4 mM MgCl2, 400 µM
dNTPs, and 1.25 U Taq Polymerase (Life Technologies) in a total
volume of 12.5 µl using 240 nM each of forward and reverse
primers (Table 1) specific for mouse GST-Ya, -Yc, and -Yp1 and
rat GST-Yb1; mouse NQO1; UDP glycosyl transferase (UGT);
GCLS; HO-1; GPx1 and 2; GR; SODs 1, 2, and 3; and catalase. 
-actin was used as an internal control. PCR was started with 5 min
incubation at 94°C followed by a three-step temperature cycle:
denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, and
extension at 72°C for 1 to ~ 2 min for 25 to ~ 30 cycles (see Table
1). A final extension step at 72°C for 10 min was included after
the final cycle to complete polymerization. The number of cycles
was chosen to ensure that amplification product did not reach a
plateau level. Reactions were electrophoresed in 2% agarose gel
containing ethidium bromide. The volume of each cDNA band
was quantitated using a Gel Doc 2000 System (Bio-Rad), and the
ratio of each gene cDNA to -actin cDNA was determined.
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Lung Cytosol Preparation
For preparation of crude cytosol, right lung tissues from four
mice of each group were pooled and homogenized in ice-cold 10 mM Tris-HCl (pH 7.8). The homogenates were centrifuged at
10,000 × g for 20 min at 4°C. Protein concentration of the resulting supernatant was determined using the Bradford assay (Bio-Rad). Aliquots of the supernatant were stored at 70°C.
Lung NQO1 Assay
Dicoumarol-sensitive NQO1 activity was measured in cytosolic fractions at 25°C by a method reported by Shaw and colleagues (28). The reaction mixture contained 30 µg cytosolic protein, 25 mM Tris-HCl (pH 7.4), 0.23 mg/ml crystalline bovine serum albumin, 0.01% (vol/vol) polyethylene sorbitan monolaurate (Tween 20, Bio-Rad), 5 µM FAD, 0.2 mM NADH, and 0 or 10 µM dicoumarol in a final volume of 200 µl. To initiate the reaction, 40 µM of 2,6-dichloroindophenol (electron acceptor) was added, and the initial velocity of the reduction of dichloroindophenol was measured spectrophotometrically at 600 nm (aM = 2.1 × 104 M/cm). The nonenzymatic rate measured in the presence of dicoumarol was subtracted from the uninhibited rate (i.e., rate in the absence of dicoumarol). Activity was measured three times, and group mean activity was expressed as nmol/min/mg protein.
Lung Total GST Assay
Total GST activity of the cytosolic preparation was measured spectrophotometrically at 25°C according to procedures published previously (29). Cytosolic protein (45 µg) was added to a 200-µl reaction mixture containing 100 mM KH2PO4 (pH 6.5) and 1 mM glutathione. Formation of the thioether between glutathione and CDNB was monitored at 340 nm (aM = 9.6 × mM/cm) by adding 1 mM CDNB to the reaction. Measurements were performed three times, and activity was expressed as nmol/min/mg protein.
Statistics
Data were expressed as the group mean ± standard error of the
mean (SEM). Three-way analysis of variance was used to evaluate the effects of hyperoxia exposure on BALF protein and cells as well as lung antioxidant enzyme mRNA expression and activity
between Nrf2 knockout (Nrf2/) and wt (Nrf2+/+) mice (n = 4 per group). The factors in the analysis were exposure (hyperoxia
or air), genotype (Nrf2/ or Nrf2+/+), and exposure time (48 or 72 h). Data sets were tested for homoscedasticity as required for parametric analyses, and data that did not meet this requirement (that
is, heteroscedastic) were natural log transformed. The Student-
Newman-Keuls test was used for a posteriori comparisons of
means. All analyses were performed using a commercial statistical analysis package (SigmaStat; Jandel Scientific Software, San
Rafael, CA). Statistical significance was accepted at P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Targeted Disruption of Nrf2 on Hyperoxia-Induced Lung Injury
The role of NRF2 in hyperoxic lung injury was evaluated by
comparing pulmonary responses to hyperoxia in Nrf2/ and
Nrf2+/+ mice. Statistically significant (P < 0.05) effects of genotype and time were detected on total protein and numbers
of macrophages and epithelial cells recovered by BALF. No
statistically significant effects of genotype, exposure, or time
were found for BALF lymphocytes or PMNs. Compared
with genotype-matched air controls, hyperoxia induced statistically significant increases in mean total protein concentration and numbers of BALF macrophages and epithelial
cells in Nrf2+/+ and Nrf2/ mice at 72 h (Figure 1). However, the mean numbers of BALF macrophages and epithelial cells were 47 and 43% greater, respectively, in Nrf2/
mice compared with those in Nrf2+/+ mice after 72 h of hyperoxia (Figure 1). Furthermore, total protein concentration
was significantly higher in Nrf2/ mice compared with
Nrf2+/+ after 48 (7.6-fold) and 72 h (3.8-fold) exposure (Figure 1). The results therefore indicate that disruption of Nrf2
significantly enhanced pulmonary sensitivity and responsivity to hyperoxic challenge.
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Effect of Hyperoxia on Lung NRF2 mRNA Expression and DNA Binding Activity
Expression levels of NRF2 mRNA were measured by Northern blot analysis to determine whether hyperoxia exposure modulates mRNA levels of NRF2. NRF2 mRNA was not detectable in the lungs of air- or hyperoxia-exposed knockout mice (Figure 2A). In contrast, constitutive expression of NRF2 mRNA (2.38 Kb) was detected in the lungs of Nrf2+/+ mice, and hyperoxia enhanced the steady-state level of NRF2 mRNA in the wt mice at 48 and 72 h (2- and 2.6-fold, respectively) compared with those in the air-exposed wt mice (Figure 2A).
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NF-E2- and ARE-binding abilities of lung nuclear proteins were assessed by EMSA to determine whether hyperoxia enhances functional NRF2 activity of Nrf2+/+ mice.
The nuclear protein-DNA complex formation in the lung
identified by shifted bands was greater in the mice exposed to 48 (2-fold) and 72 (3-fold) h of hyperoxia than in
the air-exposed mice, regardless of DNA probe used (Figure 2B). Negligible protein binding to the DNA sequences
was detected in the lungs of all Nrf2/ mice (data not
shown). To detect specific binding of NRF2 to these DNA
sequences, we performed supershift analysis using the only
commercially available anti-mouse NRF2 antibody (SC-722x; Santa Cruz Biotechnology, Santa Cruz, CA). However, we failed to obtain satisfactory supershifted bands
representing antibody-NRF2-DNA complex in the lung of
these mice, though the same antibody has yielded successful results when applied to liver tissue (42).
Differential Expression of Lung Antioxidant Defense
Enzyme mRNAs between Nrf2+/+ and Nrf2/ Mice
Expression levels of mRNA for selected antioxidant enzymes, phase 2 detoxifying enzymes, and HO-1 were compared between Nrf2+/+ and Nrf2/ mice to identify downstream genes transcriptionally activated by NRF2 (Figure
3). -Actin mRNA expression was not significantly different between genotypes or exposures (data not shown).
Hyperoxia significantly increased the mRNA expression
for NQO1 (48 and 72 h), GST-Ya (72 h), UGT (72 h),
GPx2 (48 and 72 h), and HO-1 (48 and 72 h) in the Nrf2+/+
mice over basal levels (Figure 3). The induced gene levels
of all these enzymes as well as basal mRNA levels of
NQO1 and UGT in the Nrf2+/+ mice were significantly
higher than those in the Nrf2/ animals, although UGT
and HO-1mRNAs were also inducible in the Nrf2/ mice
after hyperoxia (Figure 3). The steady-state expression level of GST-Yc mRNA was upregulated by hyperoxia exposure in the Nrf2/ mice but not in the wt mice. However, both baseline and induced mRNA levels of GST-Yc
in the wt mice were significantly greater than those in similarly exposed Nrf2/ mice (Figure 3). Northern blot analyses were used to confirm small, but statistically significant, differences in enzyme gene expression as detected by
reverse transcriptase polymerase chain reaction (RT-PCR)
(data not shown).
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The differential mRNA expression of all antioxidant
defense enzymes assessed between Nrf2+/+ and Nrf2/ mice
exposed to either hyperoxia or air are summarized in Figure 4. In addition to those discussed above, the steady-state levels of mRNAs for GCLS (48 and 72 h), GPx1 (72 h),
and GR (72 h) were markedly enhanced in the Nrf2+/+
mice by hyperoxia, whereas the mRNA level for SOD2
(48 and 72 h) was significantly elevated only in the Nrf2/
mice (see Figure 4). However, no significant differences in the abundance of these enzyme mRNAs were detected between
Nrf2+/+ and Nrf2/ mice. No statistically significant effects
of either hyperoxia or genotype were found on the mRNA
levels of GST-Yb1 and SODs 1 and 3 (see Figure 4).
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Differential Activities of Lung Phase 2 Enzymes Between
Nrf2+/+ and Nrf2/ Mice
Basal NQO1 activity was 50% greater in the lungs of
Nrf2+/+ mice compared with Nrf2/ mice (Figure 5A). Hyperoxia significantly enhanced NQO1 activity in lung cytosol
of Nrf2+/+ mice at 48 and 72 h (55 and 74%, respectively).
Hyperoxia did not change lung NQO1 activity in Nrf2/
mice. Furthermore, NQO1 activity in hyperoxia-exposed
Nrf2/ mice was significantly lower than that in similarly
exposed Nrf2+/+ mice.
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The total GST activity measured in wt mice exposed to
either air or hyperoxia (72 h) was significantly higher (1.6- to ~ 2.2-fold) than that measured in the corresponding
Nrf2/ mice (Figure 5B). No exposure-induced changes
were observed in the total GST activity in the lungs of
both genotypes of mice.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have demonstrated that NRF2 contributes to the protection against hyperoxic lung injury in mice. Compared
with wt mice, mice lacking NRF2 expression and activity
had significantly enhanced lung damage characterized by
increased protein permeability, macrophage inflammation,
and epithelial injury after hyperoxia exposure. Upregulation
of NRF2 mRNA and increased DNA binding of nuclear NRF2 was found in the lungs of wt mice in response to hyperoxia. Furthermore, significant attenuation in basal and/or
hyperoxia-induced mRNA expression of NQO1, GST-Ya
and -Yc (which compose the class 
GST in rodents), UGT,
HO-1, and GPx2 was observed in the lungs of mice deficient
in Nrf2, relative to the wt mice. This suggests that these
enzyme genes are downstream effector molecules transcriptionally activated by NRF2 in the lungs of mice. NRF2-mediated pulmonary protection against hyperoxia may be
attributed at least in part to these enzymes.
Previous studies using Nrf2-knockout mice and Nrf2-transfected or -deficient cell lines demonstrated that NRF2, in association with other transcription factors such as c-Jun and small Maf, plays an essential role in preventing carcinogenesis of cells or tissues (e.g., liver) (17, 21, 30, 31). This activity is thought to occur via ARE-mediated induction of phase 2 detoxifying enzymes including NQO1, GST, or GCLS. Chan and Kan have suggested a protective role of NRF2 against butylated hydroxytoluene (BHT) through the activation of pulmonary antioxidant defense enzymes (19). These investigators demonstrated that Nrf2-knockout mice exposed to BHT had more severe acute lung injury and lower levels of lung mRNA transcripts for antioxidant defense enzymes including NQO1, UGT, catalase, and SOD1 than similarly exposed wt mice. The potential contribution of NRF2 in oxidative tissue injury has been demonstrated in a study by Ishii and colleagues (22), who reported that peritoneal macrophages isolated from electrophile-susceptible Nrf2-knockout mice had impaired mRNA induction of HO-1, A170, and peroxiredoxin MSP23. They concluded that NRF2 is a key transcription factor for oxidative stress-inducible proteins. The present study, to our knowledge, is the first to demonstrate a protective role of NRF2 in oxidative tissue injury of the lungs.
We have determined that both basal and hyperoxia-inducible NQO1 expression are NRF2 dependent in the
murine lungs. In addition, transcriptional regulation of GST
isozyme and UGT was also at least in part mediated
through NRF2 in this model. Consistent with their NRF2-dependent gene expression patterns, enzyme activities for
lung NQO1 were significantly higher in wt mice than in
Nrf2/ mice. Total GST activity, which is attributed to all
isoenzymes (e.g., , µ, 
, and 
), was also significantly
higher in wt mice than in Nrf2/ mice following air or hyperoxia exposure. Although the total GST activity does
not discriminate between the contributions of the various
isoenzymes, results largely reflected the mRNA expression pattern of GST (composed of Ya and Yc subunits;
see Figure 3). The antioxidant role of phase 2 detoxifying
enzymes has been widely examined in cells and several tissues due to their protection against toxic and neoplastic effects of electrophilic metabolites or ROS generated by
chemical carcinogenes or xenobiotics (14, 15). Moreover,
as a component of the glutathione redox system, GST has
been postulated to provide protection to the lung from oxidative injury induced by toxicants (32). However, only
one previous study has investigated the contribution of
phase 2 enzymes to the hyperoxic lung injury in laboratory
animals (33). In that study, increased pulmonary NQO1
activity by pretreatment with 3-methylcholanthrene and
BHT did not significantly improve the survival rate of rats
exposed to hyperoxia. Our observations suggest that in addition to conjugating reactive electrophilis or xenobiotics,
phase 2 detoxifying enzymes may also exert indirect antioxidant functions in the hyperoxic lungs of mice. However, functional analyses are necessary to establish their
importance in the pathogenesis of oxidative lung injury.
Accumulating evidence has suggested that the microsomal enzyme HO-1 is highly inducible as a protective mechanism by various oxidative stresses, including hyperoxia
(13, 34) and electrophiles that induce phase 2 enzymes
(35). Recent in vitro studies (20, 36) and an in vivo study
using Nrf2 gene-knockout mice (22) have determined that
NRF2 upregulates ARE-mediated HO-1 expression. The
present study demonstrated that hyperoxia-inducible lung
HO-1 expression is partially mediated through NRF2. Interestingly, we also observed a significant induction of
HO-1 mRNA in the Nrf2/ mice by hyperoxia challenge,
which could be explained by evidence indicating that either NF-
B (37) or AP-1 (38) plays a role in the transcriptional regulation of HO-1.
Among lung classical antioxidant enzymes, regulation of mRNA for GPx2, a recently identified isoform of cellular GPxs in the gastrointestine of rodents (39), was largely NRF2-dependent in the lungs of hyperoxia-exposed mice. To date, only GPx1 has been widely investigated as the representative isoform of cellular GPx in the lungs of laboratory animals. However, a study using a mouse model with targeted disruption of GPx1 demonstrated that the hyperoxic survival rate was not increased in GPx1-deficient mice (40). The results from this and our current study suggest an important role of GPx2 as a critical component of pulmonary antioxidant defense system. We found that catalase and SOD (1, 2, and 3) mRNA expression were not dependent on NRF2 in the hyperoxic lungs. It is likely that the contribution of NRF2 to the induction of these lung antioxidant enzymes may be very limited in the protection against oxygen toxicity. These observations are inconsistent with the results from a previous study in which BHT treatment induced mRNA expression of SOD1 and catalase via NRF2 (19).
The present study also demonstrates that hyperoxia exposure enhances expression of NRF2 mRNA and functionally activated nuclear NRF2 in the lungs of normal (wt) mice. The regulatory mechanisms of NRF2 have been largely unknown with the exception of Keap1, a cytoplasmic chaperone that suppresses NRF2 transcriptional activity by specific binding to the N-terminal regulatory domain (Neh2) of NRF2 (41). Ishii and colleagues (22) postulated that NRF2 may be activated at the posttranslation level, probably by deactivation of Keap1 and, in turn, induction of NRF2 nuclear translocation. However, upregulation of the liver NRF2 mRNA level and a subsequent increase of nuclear NRF2 translocation has been reported in a recent in vivo study with mice treated with a cancer chemoprotective agent (42). This recent investigation and our present observation provide new understanding of the regulatory mechanisms of NRF2.
In conclusion, we determined that NRF2 plays a significant role in the protection against hyperoxic pulmonary injury in mice possibly by transcriptional activation of lung antioxidant defense enzymes. The results from our study add a potential protective mechanism through NRF2 and a putative role of phase 2 detoxifying enzymes as indirect antioxidants in oxidative lung injury.
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Footnotes |
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Address correspondence to: Steven R. Kleeberger, Ph.D., Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.
(Received in original form January 16, 2001 and in revised form September 17, 2001).
Abbreviations: antioxidant response element, ARE; bronchoalveolar lavage fluid, BALF; butylated hydroxyanisole, BHA; butylated hydroxytoluene, BHT; 1-chloro 2,4-dinitrobenzene, CDNB;-dithiothreitol, DTT;
ethylenediaminetetraacetic acid, EDTA; electrophoretic mobility shift assay, EMSA; flavine adenine dinucleotide, FAD; -glutamate cystein ligase
regulatory subunit, GCLS; glutathione peroxidase, GPx; glutathione reductase, GR; glutathione-S-transferase, GST; N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid, HEPES; tris buffered EDTA, TBE; heme
oxygenase-1, HO-1; 3-(N-morpholino)propanesulfonic, MOPS; nicotinamide adenine dinucleotide, reduced form, NADH; nuclear factor,
erythroid 2, NF-E2; NAD(P)H:quinone oxidoreductase 1, NQO1; NF-E2-related factor 2, NRF2; ICR/Sv129 mice, Nrf2+/+; ICR/Sv129 mice
with site-directed mutation in Nrf2, Nrf2/; polymorphonuclear leukocyte, PMN; phenylmethanesulfonyl fluoride, PMSF; polydeoxyinosinic-deoxycytidylic acid, polydI-dC; reactive oxygen species, ROS; reverse
transcriptase polymerase chain reaction, RT-PCR; standard error of the
mean, SEM; superoxide dismutase, SOD; UDP glycosyl transferase, UGT.
Acknowledgments: The authors acknowledge the contribution of the Johns Hopkins University NIEHS Center-supported Inhalation Facility to this project. This work was supported by National Institutes of Health grants ES-03819, ES-09606, HL-58122, HL-66109, HL-57142, and CA-44530 and by Environmental Protection Agency grant R-825815.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 X.-L. Chen, G. Dodd, S. Thomas, X. Zhang, M. A. Wasserman, B. H. Rovin, and C. Kunsch Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1862 - H1870. [Abstract] [Full Text] [PDF]
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M. P. Merker, S. H. Audi, R. D. Bongard, B. J. Lindemer, and G. S. Krenz Influence of pulmonary arterial endothelial cells on quinone redox status: effect of hyperoxia-induced NAD(P)H:quinone oxidoreductase 1 Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L607 - L619. [Abstract] [Full Text] [PDF]
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 P. Nioi, T. Nguyen, P. J. Sherratt, and C. B. Pickett The Carboxy-Terminal Neh3 Domain of Nrf2 Is Required for Transcriptional Activation Mol. Cell. Biol., December 15, 2005; 25(24): 10895 - 10906. [Abstract] [Full Text] [PDF]
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 T. Iizuka, Y. Ishii, K. Itoh, T. Kiwamoto, T. Kimura, Y. Matsuno, Y. Morishima, A. E. Hegab, S. Homma, A. Nomura, et al. Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema Genes Cells, December 1, 2005; 10(12): 1113 - 1125. [Abstract] [Full Text] [PDF]
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Y. Ishii, K. Itoh, Y. Morishima, T. Kimura, T. Kiwamoto, T. IIzuka, A. E. Hegab, T. Hosoya, A. Nomura, T. Sakamoto, et al. Transcription Factor Nrf2 Plays a Pivotal Role in Protection against Elastase-Induced Pulmonary Inflammation and Emphysema J. Immunol., November 15, 2005; 175(10): 6968 - 6975. [Abstract] [Full Text] [PDF]
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S. H. Audi, R. D. Bongard, G. S. Krenz, D. A. Rickaby, S. T. Haworth, J. Eisenhauer, D. L. Roerig, and M. P. Merker Effect of chronic hyperoxic exposure on duroquinone reduction in adult rat lungs Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L788 - L797. [Abstract] [Full Text] [PDF]
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S. A. Rushworth, X.-L. Chen, N. Mackman, R. M. Ogborne, and M. A. O'Connell Lipopolysaccharide-Induced Heme Oxygenase-1 Expression in Human Monocytic Cells Is Mediated via Nrf2 and Protein Kinase C J. Immunol., October 1, 2005; 175(7): 4408 - 4415. [Abstract] [Full Text] [PDF]
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H.-J. Kim and A. E. Nel The Role of Phase II Antioxidant Enzymes in Protecting Memory T Cells from Spontaneous Apoptosis in Young and Old Mice J. Immunol., September 1, 2005; 175(5): 2948 - 2959. [Abstract] [Full Text] [PDF]
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J.-M. Lee, J. Li, D. A. Johnson, T. D. Stein, A. D. Kraft, M. J. Calkins, R. J. Jakel, and J. A. Johnson Nrf2, a multi-organ protector? FASEB J, July 1, 2005; 19(9): 1061 - 1066. [Abstract] [Full Text] [PDF]
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A. Y. Shih, S. Imbeault, V. Barakauskas, H. Erb, L. Jiang, P. Li, and T. H. Murphy Induction of the Nrf2-driven Antioxidant Response Confers Neuroprotection during Mitochondrial Stress in Vivo J. Biol. Chem., June 17, 2005; 280(24): 22925 - 22936. [Abstract] [Full Text] [PDF]
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H.-Y. Cho, A. E. Jedlicka, R. Clarke, and S. R. Kleeberger Role of Toll-like receptor-4 in genetic susceptibility to lung injury induced by residual oil fly ash Physiol Genomics, June 16, 2005; 22(1): 108 - 117. [Abstract] [Full Text] [PDF]
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A. Banning, S. Deubel, D. Kluth, Z. Zhou, and R. Brigelius-Flohe The GI-GPx Gene Is a Target for Nrf2 Mol. Cell. Biol., June 15, 2005; 25(12): 4914 - 4923. [Abstract] [Full Text] [PDF]
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S. Uhlig Who Tidies Up the Lung?: Listen to Cox-2 and Nrf2 Am. J. Respir. Crit. Care Med., June 1, 2005; 171(11): 1198 - 1199. [Full Text] [PDF]
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M. Mochizuki, Y. Ishii, K. Itoh, T. Iizuka, Y. Morishima, T. Kimura, T. Kiwamoto, Y. Matsuno, A. E. Hegab, A. Nomura, et al. Role of 15-Deoxy{Delta}12,14 Prostaglandin J2 and Nrf2 Pathways in Protection against Acute Lung Injury Am. J. Respir. Crit. Care Med., June 1, 2005; 171(11): 1260 - 1266. [Abstract] [Full Text] [PDF]
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 Z. Xu, L. Chen, L. Leung, T. S. B. Yen, C. Lee, and J. Y. Chan Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia PNAS, March 15, 2005; 102(11): 4120 - 4125. [Abstract] [Full Text] [PDF]
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M. J. Calkins, R. J. Jakel, D. A. Johnson, K. Chan, Y. W. Kan, and J. A. Johnson Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription PNAS, January 4, 2005; 102(1): 244 - 249. [Abstract] [Full Text] [PDF]
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D. D. Zhang, S.-C. Lo, J. V. Cross, D. J. Templeton, and M. Hannink Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex Mol. Cell. Biol., December 15, 2004; 24(24): 10941 - 10953. [Abstract] [Full Text] [PDF]
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I. Bae, S. Fan, Q. Meng, J. K. Rih, H. J. Kim, H. J. Kang, J. Xu, I. D. Goldberg, A. K. Jaiswal, and E. M. Rosen BRCA1 Induces Antioxidant Gene Expression and Resistance to Oxidative Stress Cancer Res., November 1, 2004; 64(21): 7893 - 7909. [Abstract] [Full Text] [PDF]
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A. K. Bauer, A. M. Malkinson, and S. R. Kleeberger Susceptibility to neoplastic and non-neoplastic pulmonary diseases in mice: genetic similarities Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L685 - L703. [Abstract] [Full Text] [PDF]
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S. Papaiahgari, S. R. Kleeberger, H.-Y. Cho, D. V. Kalvakolanu, and S. P. Reddy NADPH Oxidase and ERK Signaling Regulates Hyperoxia-induced Nrf2-ARE Transcriptional Response in Pulmonary Epithelial Cells J. Biol. Chem., October 1, 2004; 279(40): 42302 - 42312. [Abstract] [Full Text] [PDF]
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K. Iida, K. Itoh, Y. Kumagai, R. Oyasu, K. Hattori, K. Kawai, T. Shimazui, H. Akaza, and M. Yamamoto Nrf2 Is Essential for the Chemopreventive Efficacy of Oltipraz against Urinary Bladder Carcinogenesis Cancer Res., September 15, 2004; 64(18): 6424 - 6431. [Abstract] [Full Text] [PDF]
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N. Li, J. Alam, M. I. Venkatesan, A. Eiguren-Fernandez, D. Schmitz, E. Di Stefano, N. Slaughter, E. Killeen, X. Wang, A. Huang, et al. Nrf2 Is a Key Transcription Factor That Regulates Antioxidant Defense in Macrophages and Epithelial Cells: Protecting against the Proinflammatory and Oxidizing Effects of Diesel Exhaust Chemicals J. Immunol., September 1, 2004; 173(5): 3467 - 3481. [Abstract] [Full Text] [PDF]
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M. McMahon, N. Thomas, K. Itoh, M. Yamamoto, and J. D. Hayes Redox-regulated Turnover of Nrf2 Is Determined by at Least Two Separate Protein Domains, the Redox-sensitive Neh2 Degron and the Redox-insensitive Neh6 Degron J. Biol. Chem., July 23, 2004; 279(30): 31556 - 31567. [Abstract] [Full Text] [PDF]
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X. Gao and P. Talalay Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage PNAS, July 13, 2004; 101(28): 10446 - 10451. [Abstract] [Full Text] [PDF]
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T. Yanagawa, K. Itoh, J. Uwayama, Y. Shibata, A. Yamaguchi, T. Sano, T. Ishii, H. Yoshida, and M. Yamamoto Nrf2 deficiency causes tooth decolourization due to iron transport disorder in enamel organ Genes Cells, July 1, 2004; 9(7): 641 - 651. [Abstract] [Full Text] [PDF]
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J.-M. Lee, K. Chan, Y. W. Kan, and J. A. Johnson Targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia PNAS, June 29, 2004; 101(26): 9751 - 9756. [Abstract] [Full Text] [PDF]
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A. Derjuga, T. S. Gourley, T. M. Holm, H. H. Q. Heng, R. A. Shivdasani, R. Ahmed, N. C. Andrews, and V. Blank Complexity of CNC Transcription Factors As Revealed by Gene Targeting of the Nrf3 Locus Mol. Cell. Biol., April 15, 2004; 24(8): 3286 - 3294. [Abstract] [Full Text] [PDF]
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J. P. Russell, J. B. Engiles, and J. L. Rothstein Proinflammatory Mediators and Genetic Background in Oncogene Mediated Tumor Progression J. Immunol., April 1, 2004; 172(7): 4059 - 4067. [Abstract] [Full Text] [PDF]
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 T. Ishii, K. Itoh, E. Ruiz, D. S. Leake, H. Unoki, M. Yamamoto, and G. E. Mann Role of Nrf2 in the Regulation of CD36 and Stress Protein Expression in Murine Macrophages: Activation by Oxidatively Modified LDL and 4-Hydroxynonenal Circ. Res., March 19, 2004; 94(5): 609 - 616. [Abstract] [Full Text] [PDF]
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 E. M. Sikorski, T. Hock, N. Hill-Kapturczak, and A. Agarwal The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury Am J Physiol Renal Physiol, March 1, 2004; 286(3): F425 - F441. [Abstract] [Full Text] [PDF]
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N. Wakabayashi, A. T. Dinkova-Kostova, W. D. Holtzclaw, M.-I. Kang, A. Kobayashi, M. Yamamoto, T. W. Kensler, and P. Talalay Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers PNAS, February 17, 2004; 101(7): 2040 - 2045. [Abstract] [Full Text] [PDF]
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S. Gebel, B. Gerstmayer, A. Bosio, H.-J. Haussmann, E. V. Miert, and T. Muller Gene expression profiling in respiratory tissues from rats exposed to mainstream cigarette smoke Carcinogenesis, February 1, 2004; 25(2): 169 - 178. [Abstract] [Full Text] [PDF]
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K. Itoh, M. Mochizuki, Y. Ishii, T. Ishii, T. Shibata, Y. Kawamoto, V. Kelly, K. Sekizawa, K. Uchida, and M. Yamamoto Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-{Delta}12,14-Prostaglandin J2 Mol. Cell. Biol., January 1, 2004; 24(1): 36 - 45. [Abstract] [Full Text] [PDF]
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L. Leung, M. Kwong, S. Hou, C. Lee, and J. Y. Chan Deficiency of the Nrf1 and Nrf2 Transcription Factors Results in Early Embryonic Lethality and Severe Oxidative Stress J. Biol. Chem., November 28, 2003; 278(48): 48021 - 48029. [Abstract] [Full Text] [PDF]
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J.-M. Lee, A. Y. Shih, T. H. Murphy, and J. A. Johnson NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons J. Biol. Chem., September 26, 2003; 278(39): 37948 - 37956. [Abstract] [Full Text] [PDF]
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 C. J. Henderson and C. R. Wolf Transgenic Analysis of Human Drug-Metabolizing Enzymes: Preclinical Drug Development and Toxicology Mol. Interv., September 1, 2003; 3(6): 331 - 343. [Abstract] [Full Text] [PDF]
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N. R. Hackett, A. Heguy, B.-G. Harvey, T. P. O'Connor, K. Luettich, D. B. Flieder, R. Kaplan, and R. G. Crystal Variability of Antioxidant-Related Gene Expression in the Airway Epithelium of Cigarette Smokers Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 331 - 343. [Abstract] [Full Text] [PDF]
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A. Baulig, M. Garlatti, V. Bonvallot, A. Marchand, R. Barouki, F. Marano, and A. Baeza-Squiban Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L671 - L679. [Abstract] [Full Text] [PDF]
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S. Perkowski, J. Sun, S. Singhal, J. Santiago, G. D. Leikauf, and S. M. Albelda Gene Expression Profiling of the Early Pulmonary Response to Hyperoxia in Mice Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 682 - 696. [Abstract] [Full Text] [PDF]
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 A. Y. Shih, D. A. Johnson, G. Wong, A. D. Kraft, L. Jiang, H. Erb, J. A. Johnson, and T. H. Murphy Coordinate Regulation of Glutathione Biosynthesis and Release by Nrf2-Expressing Glia Potently Protects Neurons from Oxidative Stress J. Neurosci., April 15, 2003; 23(8): 3394 - 3406. [Abstract] [Full Text] [PDF]
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M. Ramos-Gomez, P. M. Dolan, K. Itoh, M. Yamamoto, and T. W. Kensler Interactive effects of nrf2 genotype and oltipraz on benzo[a]pyrene-DNA adducts and tumor yield in mice Carcinogenesis, March 1, 2003; 24(3): 461 - 467. [Abstract] [Full Text] [PDF]
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Y.-S. Ho Transgenic and Knockout Models for Studying the Role of Lung Antioxidant Enzymes in Defense against Hyperoxia Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): S51 - 56. [Abstract] [Full Text] [PDF]
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S. P. M. Reddy and B. T. Mossman Role and regulation of activator protein-1 in toxicant-induced responses of the lung Am J Physiol Lung Cell Mol Physiol, December 1, 2002; 283(6): L1161 - L1178. [Abstract] [Full Text] [PDF]
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L. M. Zipper and R. T. Mulcahy The Keap1 BTB/POZ Dimerization Function Is Required to Sequester Nrf2 in Cytoplasm J. Biol. Chem., September 20, 2002; 277(39): 36544 - 36552. [Abstract] [Full Text] [PDF]
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A. T. Dinkova-Kostova, W. D. Holtzclaw, R. N. Cole, K. Itoh, N. Wakabayashi, Y. Katoh, M. Yamamoto, and P. Talalay Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants PNAS, September 3, 2002; 99(18): 11908 - 11913. [Abstract] [Full Text] [PDF]
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M.-K. Kwak, K. Itoh, M. Yamamoto, and T. W. Kensler Enhanced Expression of the Transcription Factor Nrf2 by Cancer Chemopreventive Agents: Role of Antioxidant Response Element-Like Sequences in the nrf2 Promoter Mol. Cell. Biol., May 1, 2002; 22(9): 2883 - 2892. [Abstract] [Full Text] [PDF]
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L. E. Otterbein and A. M. K. Choi The Saga of Leucine Zippers Continues . In Response to Oxidative Stress Am. J. Respir. Cell Mol. Biol., February 1, 2002; 26(2): 161 - 163. [Full Text] [PDF]
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