| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*
Division of Clinical Immunology and Allergy, Department of Medicine, School of Medicine;
Institute of Geophysics and Planetary Physics; and
Southern California Particle Center and Supersite, University of California, Los Angeles, CA 90095; and
§
Division of Research, Alton Ochsner Medical Foundation, New Orleans, LA 70121
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The ARE, with underlined consensus sequence GTGACNNNGC, is a transcriptional regulatory element that plays a role in the expression of phase II enzymes such as glutathione-S-transferase, NAD(P)H:quinone oxidoreductase, and glucuronosyltransferase (9, 10, 11, 12, 13, 14). The ability of these enzymes to conjugate redox-cycling chemicals is an important protective mechanism against electrophile and oxidative toxicity. Prototypical chemicals are phenolic antioxidants, Michael reaction acceptors, isothiocyanates, trivalent arsenicals, and redox-cycling polycyclic aromatic hydrocarbons (PAH) and quinones (10, 11, 12, 13, 14, 15, 16, 17). Although this antioxidant defense mechanism has been studied extensively as a hepatic detoxification mechanism, it has also been suggested that the ARE pathway may contribute to antioxidant defenses in the lung (18). An example is regulation of heme oxygenase-1 (HO-1) expression by the SX2 enhancer, a 268-bp gene fragment that is located 4 kb upstream of the start site (18, 19, 20). This enhancer contains two AREs and can be activated by some of the chemical species listed above (20). Because DEP contain a variety of organic substances that undergo redox cycling (21, 22), we were interested in whether DEP extracts may induce HO-1 expression (23). HO-1 confers protection against oxidative lung injury (24, 25) and may therefore play a role in the pulmonary defense against particulate pollutants. We were particularly interested in the role of oxygenated PAH such as quinones in this pathway, because these chemicals have been implicated in ROS generation by DEP extracts in lung microsomal preparations (24, 25).
HO-1 protein expression can be induced throughout the lung, with particular abundance in the alveolar and bronchiolar epithelium as well as airway inflammatory cells, including macrophages (24, 25). Although bronchoalveolar macrophages have an important role in DEP clearance from the airway (5), macrophage activation by DEP may contribute to the pulmonary inflammation and enhanced IgE production attributed to this particulate matter (PM) (1, 26). Because the ARE pathway is operational in macrophages, it is possible that this pathway may play a role in the defense against DEP chemicals, including PAH and quinones (27). In this regard we have previously shown that b-napthoflavone (a PAH) and tert-butylhydroquinone (tBHQ; a quinone and a phenolic antioxidant) induce ARE activation in macrophages (27).
We wished to determine whether DEP chemicals, including PAH and quinone fractions, induce antioxidant defenses through an effect on HO-1 expression in macrophages. We demonstrate that DEP total extracts, polar and aromatic DEP fractions, as well as benzo(a)pyrene-3,6-quinone (BPQ) induce HO-1 expression in parallel with transcriptional activation of AREs in the HO-1 enhancer. These events could be suppressed by N-acetyl-cysteine (NAC) or mutagenesis of the AREs in the SX2 enhancer. This establishes a biologically relevant role for the ARE in antioxidant defenses in macrophages.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DMEM, macrophage serum-free medium, penicillin-streptomycin, L-glutamine, and FBS were purchased from Life Technologies (Gaithersburg, MD). DEP were a gift from Dr. Masaru Sagai (National Institute of Environment Studies, Tsukuba, Ibaraki, Japan). Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) was a gift from Dr. Judith Berliner (University of California, Los Angeles, CA). NAC, tBHQ, propidium iodide (PI), silibinin, and LPS were obtained from Sigma (St. Louis, MO). Anti-HO-1 mAb was purchased from Stressgen (Victoria, Canada). Sheep anti-mouse Ab was obtained from Amersham (Arlington Heights, IL). A luciferase assay kit was purchased from PharMingen (San Diego, CA). Enhanced chemiluminescence reagents were obtained from Pierce (Rockford, IL). An endotoxin-free Maxiprep kit was purchased from Promega (Madison, WI). TRIzol RNA extraction reagent, reverse transcriptase, and Taq DNA polymerase were obtained from Life Technologies. Dichlorofluorescein diacetate (DCF) was purchased from Molecular Probes (Eugene, OR). Luteolin was obtained from Calbiochem (La Jolla, CA). Cobalt protoporphyrin (CoPP) was purchased from Porphyrin Products (Logan, UT).
Cell culture and transfection
RAW264.7 were cultured as previously described (6).
Plasmid DNA was prepared using the endotoxin-free Maxiprep kit
according to the manufacturer’s instructions (Promega). The sequences
of the wild-type and mutant SX2 elements (SX2, M2, and M239) in the
5'-upstream region of the mouse HO-1 gene have been described
previously (18, 19, 20) (Fig. 1
). These enhancer elements were
subcloned into the pD44-luciferase plasmid vector. Transfections were
conducted using previously described methods (27).
Briefly, 7 x 106 RAW264.7 cells resuspended
in DMEM containing 20% FBS were transfected with 20 µg of the
reporter cDNA using an electroporator (Bio-Rad, Hercules, CA) with
voltage set at 260 V and capacitance set at 975 µF. Cells were
resuspended in DMEM containing 10% FBS and were rested for 24 h
before the addition of various stimuli as shown in the figure
legends.
|
The tBHQ, BPQ, silibinin, and luteolin stock solutions were prepared in DMSO. DEP extracts were prepared as previously described (6). Briefly, 100 mg DEP was suspended in 25 ml of methanol and sonicated for 30 s. The DEP/methanol suspension was centrifuged at 2000 rpm for 10 min at 4°C. The methanol supernatant was transferred to a preweighed polypropylene tube and dried under nitrogen gas (6). Dried DEP extracts were resuspended in DMSO at 100 mg/ml and stored at -20°C in the dark. The NAC stock solution (1 M) was made in HEPES immediately before use and was diluted to a final concentration of 20 mM in culture medium. The antioxidants were added 2 h before and during cellular stimulation. CoPP stock (25 µg/µl) was made in 0.1 M NaOH and stored at -80°C. For HO-1 induction, CoPP was diluted in the culture medium to the desired concentrations, after which the pH was adjusted, and the medium was sterile-filtered. An equal amount of NaOH was added to the control medium. OxPAPC in chloroform (2 mg/ml) was stored at -80°C, dried under nitrogen gas, and resuspended in culture medium immediately before use. RAW264.7 cells were plated at 106 cells/well in 3 ml of culture medium for 24 h before the addition of various stimuli. Controls were treated with DMSO at a final concentration of 0.1%.
Western blotting to determine HO-1 expression
The cells were harvested by scraping and were lysed as previously described (6). Fifty micrograms of total lysate protein was electrophoresed on SDS-polyacrylamide gels before transfer to nitrocellulose membranes. Blots were sequentially overlayed with anti-HO-1 mAb at 0.3 mg/ml and sheep anti-mouse Ab conjugated to peroxidase according to the manufacturer’s instruction. Blots were developed with the enhanced chemiluminescence reagent according to the manufacturer’s instructions.
RT-PCR analysis
Total RNA was extracted using TRIzol RNA extraction reagent (1). RT was conducted at 42°C for 1 h in a total volume of 20 µl containing 5 µg of total RNA; 0.5 mg of oligo(dT)12–18; 10 mM DTT; 0.5 mM each of dATP, dGTP, dCTP, and dTTP; and 10 U of Superscript II reverse transcriptase. HO-1 primers for PCR amplification of a 668-bp fragment from exon 1 of mouse HO-1 gene (28) were obtained from Life Technologies. The forward (sense) primer was 5'-CTGTGTAACCTCTGCTGTTCC-3', and the reverse (antisense) primer was 5'-CCACACTACCTGAGTCTACC-3'. Primers for mouse ß-actin mRNA were used as an internal control. The forward (sense) primer was 5'-TGGAATCCTGTGGCATCCATGAAAC-3', and the reverse (antisense) primer was 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'. PCR was performed in a total volume of 25 µl, using 4 µl of cDNA template, 0.5 mM of the sense and antisense primers, 2.5 U of Taq DNA polymerase, and 2 mM MgCl2 in a Perkin-Elmer thermocycler (Norwalk, CT). After amplification for 35 cycles (1 min at 94°C, 1 min at 55°C, and 1 min at 72°C), PCR products were electrophoresed in 2% agarose gels and viewed by ethidium bromide staining.
Luciferase (Luc) assay
Transfected cells were collected by scraping and were lysed with the luciferase lysis buffer according to the manufacturer’s instruction. Luc assays were performed on 50 mg lysate protein, using the manufacturer’s assay kit and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).
DEP fractionation and gas chromatography/mass spectrometry (GC/MS) analysis
One gram of DEP was extracted with 60 ml of methylene chloride five times using a Vir-Tis homogenizer (Virtis, Gardiner, NY). The combined extracts were concentrated by rotoevaporation, and asphaltenes were precipitated by exchange into hexane. The supernatant was concentrated, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (column size, 1 x 30 cm) following the method of Venkatesan et al. (29). Aliphatic, aromatic, and polar fractions were collected by elution with 20 ml of hexane, 40 ml of hexane/methylene chloride (3/2), and 30 ml of methylene chloride/methanol (1/1), respectively. The fractions were weighed in a microbalance by evaporating off a known volume of an aliquot of the sample made up in methylene chloride or methanol.
GC with Flame ionization detector (Varian 3400 GC with a SPI injector and a DB-5 column, 30 m, 0.25 mm id, 0.25 µm film; Varian, Walnut Creek, CA) was used to chemically characterize the alkane fraction. The aromatic and polar fractions were analyzed by a gas chromatograph/mass spectrometer (Finnigan 4000 (Finnigan-MAT, San Jose, CA) equipped with a Varian 3400 GC with a split/splitless injector and Galaxy 2000 for Windows data system; Los Gatos Circuits, Los Gatos, CA). The PAHs were quantitated by GC/MS in the selective ion monitoring (SIM) mode using deuterated internal standard mixtures, and an electron energy of 70 eV was used. The mass spectrometer was operated on SIM mode, using appropriate optimum windows to include the quantitation and confirmation masses for the analytes. A five-point response factor calibration curve was established. Standard concentrations used to construct the calibration curve were 1, 5, 10, 20, and 50 ng/µl. The identification of compounds detected at concentration above the method detection limit (MDL) was double checked by the confirmation ions. If the concentration of the target analyte exceeded the linear range of the calibration standards, the fraction was either concentrated or diluted and reanalyzed. The polar fraction was taken up in dichloromethane/acetonitrile (1/1, v/v) and analyzed by GC-MS in the SIM mode and the SCAN mode to search for oxy-PAHs, quinones, and ketones using total ion count spectra and the National Institute of Standards and Technology library.
Flow cytometric analysis
ROS generation was determined by DCF fluorescence, and cell viability was determined by PI uptake as previously described (6). Briefly, cells were incubated in 2.5 µM DCF in DMEM (106/ml) at 37°C in the dark for 30 min. PI (2.5 µg/ml) was added before the analysis. For viability analysis to study the effect of CoPP, cells were stained with PI (2.5 µg/ml) for 15 min at room temperature in the dark. Flow cytometry was performed using a FACScan equipped with an argon laser (Becton Dickinson, Franklin Lakes, NJ). The mean fluorescence intensities for DCF and PI were analyzed in FL-1 and FL-2 channels, respectively.
Statistics
Data were analyzed using SAS statistical software (SAS Institute, Cary, NC). Scheffe’s method of multiple comparisons with F test was used for ANOVA.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A variety of stimuli can induce HO-1 expression both in vitro and
in vivo. These factors include bacterial endotoxin (LPS), IL-1,
hyperoxia, heavy metals, as well as some electrophilic phase 2 enzyme
inducers (18, 30, 31). Moreover, Prestera et al. reported
that a prototypic phase II enzyme inducer, tBHQ, induces HO-1 gene
expression in mouse hepatoma cells (20). We tested the
same chemical in RAW264.7 cells. Following incubation with tBHQ for
16 h, RAW264.7 cells showed HO-1 expression, as determined by
immunoblotting (Fig. 2A).
Inclusion of NAC in the culture medium significantly reduced HO-1
expression (Fig. 2A). Treatment of RAW264.7 cells with tBHQ
also induced HO-1 mRNA expression, as determined by RT-PCR analysis
(Fig. 2B).
|
) (20). We transfected RAW264.7 cells with
the SX2 fragment linked to a Luc reporter. Subsequent treatment with
tBHQ induced a 4-fold increase in SX2-Luc activity (Fig. 3A). For protein expression,
this response was significantly suppressed by 20 mM NAC (Fig. 2A).
|
) (18, 19, 20). Our results demonstrate a significant
reduction in the SX2-M239-Luc response to tBHQ treatment compared with
the wild-type reporter (Fig. 3B). Because these AREs contain
an overlapping AP-1 nucleotide sequence (GTGACTCAGC) that putatively
plays a role in regulating transcriptional activity, we compared the
effect of tBHQ on the SX2-M2-Luc mutant that eliminates the AP-1
binding site but leaves the ARE core sequence (GTGACTCAGC) intact (Fig. 1). This mutation did not interfere with the transcriptional activation
of the SX2 enhancer (Fig. 3B). Induction of HO-1 expression and promoter activity by organic DEP extracts is sensitive to the inhibitory effects of NAC and flavonoid antioxidants
Because DEP contain a wide variety chemicals that may induce
oxidative stress (6), including quinones and ketones
(22, 23), we asked whether a methanol extract made from
these particles could induce HO-1 expression in RAW264.7 cells.
RAW264.7 cells were incubated with these extracts after they were dried
and resuspended in DMSO (6). Western blot analysis showed
a dose-dependent increase in HO-1 expression (Fig. 4A), while RT-PCR analysis
showed induction of HO-1 mRNA expression by the DEP extract (Fig. 4B).
|
A).
This effect may be explained by the role of NAC as an antioxidant or
its ability to directly complex to chemical groups such as quinones
(34). Because a DEP extract is a known inducer of ROS that
can be suppressed by NAC in macrophages (6), we asked
whether other classes of antioxidants interfere with HO-1 expression.
For that purpose, we employed two flavonoid antioxidants, silibinin and
luteolin (35, 36). Although silibinin had minimal
inhibitory effects on enhanced DCF fluorescence, luteolin led to
substantive inhibition of DEP-induced ROS generation at all
concentrations tested (Fig. 5A). When
used in an assay for HO-1 induction, luteolin, but not silibinin,
interfered with HO-1 expression in a dose-dependent fashion (Fig. 5B).
Taken together, these data show that oxidative stress is involved in
DEP-induced HO-1 expression.
|
A). Thus, while a dose of
12.5 µg/ml had a slight effect, doses of 25 and 50 µg/ml induced
progressively greater responses. These concentrations are below the DEP
extract doses leading to apoptosis, i.e., concentrations >50 µg/ml
(6). Prior addition of NAC significantly reduced SX2
activation (Fig. 6A). Mutation of the core AREs (i.e., using
SX2-M239-Luc), but not the overlapping AP-1 site, resulted in a
significant decrease in reporter gene activity (Fig. 6B).
Taken together, these results show that DEP chemicals play a role in
HO-1 expression via stimulation of AREs in the SX2 domain of that
gene.
|
Previous studies of the effect of extractable diesel chemicals on
ROS generation in the lung have suggested that redox cycling quinones
are responsible for that effect (22, 23). Because this
result was based on interference of NaBH4 in the
activity of a crude methanol extract, we separated a methylene chloride
extract obtained from DEP into three fractions using silica gel
chromatography (Table I). The relative
abundance of aliphatic, aromatic, and polar components is shown in
Table I. The GC analysis of aliphatic fraction showed the presence of
mainly normal alkanes, ranging in carbon number from 13 to 31 with
C17 to C24 homologues
predominating (not shown). The PAHs, which were present in the aromatic
fraction, were characterized and quantitated by GC/MS and are listed in
Table II. Although the polar fraction did
not contain any measurable PAHs, oxygenated compounds, such as
9-H-fluorene-9-one, anthracenone/phenanthrenol, and
9,10-anthracenedione, were identified in this fraction (Table III). Using the aromatic and polar
fractions, we were able to elicit dose-dependent HO-1 expression in
RAW264.7 cells (Fig. 7). The polar
fraction was more potent than the aromatic fractions and induced a
response at 1 µg/ml (Fig. 7). This is considerably less than the
amount of crude DEP extract required for response induction, i.e., 25
µg/ml or more (Fig. 4A). The aliphatic fractions had no
discernable effect on HO-1 expression (Fig. 7).
|
|
|
|
The increased potency of the polar fraction on HO-1 expression
suggests that oxygenated PAHs such as quinones are involved. To further
explore the role of quinones, we looked at the effect of a frequently
studied quinone that is present in second-hand smoke and in PM, albeit
in minor amounts (23, 37). Oxidation of the BaP ring
yields three species, namely 1,6-, 3,6-, and 6,12-BPQ
(23). We asked whether these quinones participate in HO-1
expression in RAW264.7 cells. Exposure to 20 µM of each of these
quinones showed that while 3,6-BPQ was an effective inducer of HO-1
protein expression, the 1,6- and 6,12-quinones failed to do so (Fig. 8A). 3,6-BPQ also induced HO-1
mRNA expression, as determined by RT-PCR analysis (Fig. 4B).
HO-1 induction by this quinone was completely abolished by 20 mM NAC
(Fig. 8A). Using the SX2-Luc promoter, we could elicit a
small (2.5-fold), but statistically significant, increase in luciferase
activity with the 3,6-BPQ, but not the 1,6- or 6,12-BPQ, species (not
shown).
|
B).
Note that the basal level of HO-1 expression is increased under
serum-free conditions. This may be due to the depletion of antioxidants
from the culture medium, or it may reflect cellular stress of serum
depletion. Evidence that HO-1 has cytoprotective effects in RAW264.7 cells
There is abundant evidence that HO-1 is a stress protein with
cytoprotective effects, including pulmonary defense against LPS and
hyperoxia. To determine whether HO-1 was able to protect RAW264.7
against the cytotoxic effects of DEP extracts, these cells were
previously incubated with CoPP, an inducer of HO-1 activity. CoPP has
been shown to exert cytoprotective effects in vivo in a rat liver
transplantation model (38). Western blotting showed that
CoPP induced HO-1 expression at a dose 
7.5 µg/ml (Fig. 9A). The lower effective
concentration was used to treat RAW264.7 cells for 48 h before
treatment with 100 µg/ml of a crude DEP extract. We have previously
shown that this dose induces apoptosis in an oxidative stress-dependent
fashion (6). Using flow cytometric analysis to detect PI
uptake in dead cells, we showed that the DEP extract induced cell death
in >90% RAW264.7 cells (Fig. 9B). NAC interfered with this
event (22% dead cells; Fig. 9B). In cells pretreated with
CoPP, the rate of cell death fell to 54% (Fig. 9B),
suggesting that under conditions of HO-1 expression, cells are
protected against the toxicity of DEP chemicals.
|
In addition to chemical agents, it has been demonstrated that some
biological agents, such as bacterial LPS, could induce human ARE
reporter activity and HO-1 expression in RAW264.7 cells (18, 31). This effect may be relevant to pulmonary protection against
inhaled bacterial endotoxin as well as during Gram-negative sepsis
(39). Another biological agent that induces HO-1
expression in endothelial cells and smooth muscle cells and that has
relevance to macrophages is oxidized LDL (40).
Oxidized LDL as well as one of the phospholipids contained
therein, PAPC, has stimulatory effects on macrophages (35, 36). We asked, therefore, whether OxPAPC could induce HO-1
expression in RAW264.7 cells. OxPAPC could induce
HO-1 expression as determined by immunoblotting (Fig. 10A). RT-PCR analysis demonstrated that
RAW264.7 stimulation with OxPAPC increased HO-1 mRNA expression (Fig. 10B). This response was suppressed by 20 mM NAC (Fig. 10A). Using the SX2-Luc reporter, we could demonstrate that
OxPAPC induced robust enhancer activity in an NAC-sensitive manner
(Fig. 10C). Taken together, these data closely resemble the
effect of oxidant chemicals on HO-1 expression in RAW264.7 cells.
OxPAPC should therefore be included in the growing list of
biological agents that impact the HO-1 antioxidant defense pathway.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The ARE is a transcriptional regulatory element that is a widely recognized protector against chemical-induced electrophile and oxidative toxicity (9, 10, 11, 12, 13, 14, 15, 16, 17). This is accomplished through the expression of phase II conjugating enzymes, e.g., NADPH:quinone oxidoreductase. This enzyme performs two-electron reductions on quinones and converts them to hydroxyl quinones, which are no longer able to participate in one-electron reductions or redox-cycling events (41). Phase II enzymes therefore effectively remove the source of the electrophile and ROS, which are also the primary inducers of ARE activity. This induction of ARE activity involves activation or intracellular translocation of a constitutively expressed transcription factor in hepatocytes and macrophages (11, 27). Recently, it has been shown that members of the Nrf transcription factor family interact with the ARE (42, 43, 44), and that Nrf2 regulates induction of the HO-1 gene (45). On exposure to electrophilic agents, the DNA-binding activity of Nrf2 is markedly induced, while the Nrf2 steady state mRNA levels remain constant (45, 46). This suggests that signals from oxidizing agents are transduced from a cellular sensor to Nrf proteins. Nrf2 contains a negative regulatory domain that interacts with a novel cytoplasmic protein, Keap1 (46). Keap1 is liberated from Nrf2 in the presence of electrophilic agents (46). That allows Nrf2 to physically recompartmentalize from the cytoplasm to the nucleus. Interestingly, it was shown that the induction of a repertoire of oxidative stress genes is severely impaired in Nrf2-deficient macrophages (46).
Although the initial interest in HO-1 focused on its role in heme
catabolism, current studies show that HO-1 is highly responsive to
oxidative stress and has potent antioxidant properties
(31). Agents leading to HO-1 induction include
H2O2, UV irradiation,
sodium arsenite, heavy metals, isothiocyanates, phenolic antioxidants,
and Michael reaction acceptors (10, 11, 12, 13, 14, 15, 16, 17, 32). Due to its
antioxidant properties, HO-1 exerts cytoprotective effects in tissues
where it is expressed, including the lung (24, 25, 31).
This is in keeping with the finding that HO-1-deficient mice have a
reduced defense against chemical-induced oxidative stress, while
HO-1-deficient humans also exhibit enhanced endothelial cell injury in
the presence of oxidative stress (33, 47). Although the
expression of the HO-1 gene depends on a variety of
cis-acting elements, including heat shock, NF-
B, AP-1,
and heme response elements, oxidative chemicals operate via the AREs in
the distal enhancer (31). This suggests that, similar to
phase II enzymes, the antioxidant effect of HO-1 is regulated via
electrophile response elements. In this communication we show that the
ARE pathway is involved in HO-1 expression during RAW264.7 cell
exposure to tBHQ, DEP extracts, aromatic and polar fractions, as well
as 3,6-BPQ (Figs. 2A, 4A, and 7A).
Although mutation of the ARE core (i.e., the SX2-M239 mutant)
interfered with transcriptional activation of the SX2 enhancer,
mutational alteration of the overlapping AP-1 sequence (i.e., the
SX2-M2 mutant) had no effect on the response to the chemicals (Figs. 3B and 4D). Interestingly, the SX2-M2 mutation
did interfere with LPS responsiveness in hepatocytes, suggesting that
the latter pathway uses Fos and Jun protein rather than ARE-specific
transcription factors (18). This finding also corroborates
our previous demonstration that regardless of its ability to bind to
AP-1 proteins in vitro, the human ARE can be activated independently of
the AP-1 site in macrophages (27).
HO-1 induction appears to be part of a generalized cellular response
that protects cells against oxidative stress and other toxic insults
(32, 33). We are particularly interested in its protective
role in the lung against particulate pollutants. HO-1 expression has
been shown to play a role in protecting the rodent lung from
endotoxin-induced injury as well as the effects of hyperoxia (24, 25, 31). By using CoPP as an HO-1 inducer, we also showed that
HO-1 expression has a protective effect in macrophages (Fig. 9). Along
similar lines, we propose that HO-1 plays a protective role against the
oxidative effects of organic DEP chemicals in the lung. Among the cells
targeted by oxidative stress, we have focused on macrophages, because
these cells make the primary contact with DEP. The same principles may
also apply to epithelial and endothelial cells, which are the other
pulmonary targets for PM (48). Both cell types exhibit
HO-1 expression.
Among the >400 chemicals in DEP, we are particularly interested in the
roles of oxygenated PAH and quinones in ARE activation
(23). Kumagai et al. have shown that DEP extracts induce
O2- production in lung
microsomes in a cytochrome P450 reductase-dependent manner
(22). The same group has also provided preliminary
evidence that quinones are responsible for this oxidizing effect
(22). PAH-derived quinones are abundant in all burnt
organic matters, including automobile exhaust, cigarette smoke, and
urban air particulates (23, 49, 50). It is relevant,
therefore, that the aromatic and the polar DEP fractions,
which were shown to contain PAHs and oxygenated PAH, respectively, were
potent HO-1 inducers (Tables II and III and Fig. 7). The idea that
quinones are involved in this cellular event is also strengthened by
the finding that 3,6-BPQ could induce HO-1 expression in macrophages
(Fig. 8). BPQs undergo one-electron reductions to semiquinones by
NADPH-cytochrome P450 reductase, leading to the formation of
O2- and, subsequently,
H2O2 (37). The
same quinone isomer (3,6-BPQ), leading to HO-1 expression, is also more
mutagenic in the Ames test; mutagenesis is dependent on oxidative DNA
damage in Salmonella (37). The involvement of
oxidative stress in DEP-induced HO-1 expression was confirmed by DCF
fluorescence and the ability of NAC and luteolin to interfere with HO-1
expression (Figs. 5 and 8A). Although the effect of NAC can
be explained both through its antioxidative effects as well as by its
directly complexing to quinones, it is relevant that luteolin also
suppressed ROS production and HO-1 expression (Fig. 5). That
strengthens the idea that oxidative stress is involved in this event.
Interestingly, another flavonoid, silibinin, was ineffective in
suppressing HO-1 induction (Fig. 5B). This may be related to
the fact that this agent is a good scavenger for hypochloric acid
(HOCl), but not of O2-
(51). Taken together, we propose that oxygenated PAH and
quinones are strong candidates responsible for the observed HO-1
expression induced by the DEP extracts.
The role of the ARE in macrophages is not confined to protection
against chemical effects alone. In this regard, mildly oxidized LDL and
a phospholipid component of LDL, OxPAPC, also induced HO-1 expression
in RAW264.7 cells (40). Moreover, it has been reported
that HO-1 is induced in vascular endothelial and smooth muscle cells by
oxidized LDL in parallel with other stress proteins (40, 52). These results suggest that HO-1 induction by OxPAPC may
have a protective effect against arterial wall inflammation, such as
that seen in atherosclerosis. This protective effect may also apply to
other forms of inflammation where IL-1 and TNF-
come
into play, because it has been demonstrated that these cytokines induce
HO-1 expression (53, 54). The ARE may therefore have
biological functions beyond the detoxification of chemicals.
In summary, we show that DEP chemicals and quinones induce an antioxidant defense pathway in macrophages through an ROS-mediated effect on the ARE. Because this effect appears to be induced at lower chemical concentrations than those causing cytotoxic effects, the ARE pathway could theoretically be exploited to enhance antioxidant defenses against PM.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Andre E. Nel, Department of Medicine, Division of Clinical Immunology and Allergy, 10833 Le Conte Avenue, 52-175 CHS, Los Angeles, CA 90095.
3 Abbreviations used in this paper: DEP, diesel exhaust particles; ARE, antioxidant response element; BPQ, benzo(a)pyrene quinone; CoPP, cobalt protoporphyrin; DCF, dichlorofluorescein diacetate; GC/MS, gas chromatography/mass spectrometry; HO-1, heme oxygenase-1; Luc, luciferase; NAC, N-acetyl-cysteine; OxPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PAPC, 1-palmitoly-2-arachidonoyl-sn-glycero-3-phosphocholine; PAH, polycyclic aromatic hydrocarbon; PI, propidium iodide; PM, particulate matter; ROS, reactive oxygen species; SIM, single ion monitoring; tBHQ, tert-butylhydroquinone.
Received for publication January 4, 2000. Accepted for publication June 29, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element. Biochem. Biophys. Res. Commun. 261:661.[Medline]
and interleukin-1 on heme oxygenase-1 expression in human endothelial cells. Am. J. Physiol. 274:H883.
and IL-1 induce hemeoxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells. Am. J. Physiol. 276:H1493.This article has been cited by other articles:
|
|
 |
|
  T. Stevens, S.-H. Cho, W. P. Linak, and M. I. Gilmour Differential Potentiation of Allergic Lung Disease in Mice Exposed to Chemically Distinct Diesel Samples Toxicol. Sci., February 1, 2009; 107(2): 522 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Soberanes, D. Urich, C. M. Baker, Z. Burgess, S. E. Chiarella, E. L. Bell, A. J. Ghio, A. De Vizcaya-Ruiz, J. Liu, K. M. Ridge, et al. Mitochondrial Complex III-generated Oxidants Activate ASK1 and JNK to Induce Alveolar Epithelial Cell Death following Exposure to Particulate Matter Air Pollution J. Biol. Chem., January 23, 2009; 284(4): 2176 - 2186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Williams, T. Rangasamy, S. M. Bauer, S. Killedar, M. Karp, T. W. Kensler, M. Yamamoto, P. Breysse, S. Biswal, and S. N. Georas Disruption of the Transcription Factor Nrf2 Promotes Pro-Oxidative Dendritic Cells That Stimulate Th2-Like Immunoresponsiveness upon Activation by Ambient Particulate Matter J. Immunol., October 1, 2008; 181(7): 4545 - 4559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Rouse, G. Murphy, M. J. Boudreaux, D. B. Paulsen, and A. L. Penn Soot Nanoparticles Promote Biotransformation, Oxidative Stress, and Inflammation in Murine Lungs Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 198 - 207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Stevens, Q. T. Krantz, W. P. Linak, S. Hester, and M. I. Gilmour Increased Transcription of Immune and Metabolic Pathways in Naive and Allergic Mice Exposed to Diesel Exhaust Toxicol. Sci., April 1, 2008; 102(2): 359 - 370. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshida and R. M. Tuder Pathobiology of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease Physiol Rev, July 1, 2007; 87(3): 1047 - 1082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M. Gharavi, P. S. Gargalovic, I. Chang, J. A. Araujo, M. J. Clark, W. L. Szeto, A. D. Watson, A. J. Lusis, and J. A. Berliner High-Density Lipoprotein Modulates Oxidized Phospholipid Signaling in Human Endothelial Cells From Proinflammatory to Anti-inflammatory Arterioscler Thromb Vasc Biol, June 1, 2007; 27(6): 1346 - 1353. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Li, W. Chen, R. Yanes, S. Lee, and J. A. Berliner OKL38 is an oxidative stress response gene stimulated by oxidized phospholipids J. Lipid Res., March 1, 2007; 48(3): 709 - 715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Inoue, H. Takano, K. Hiyoshi, T. Ichinose, K. Sadakane, R. Yanagisawa, S. Tomura, and Y. Kumagai Naphthoquinone enhances antigen-related airway inflammation in mice Eur. Respir. J., February 1, 2007; 29(2): 259 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Ritz, J. Wan, and D. Diaz-Sanchez Sulforaphane-stimulated phase II enzyme induction inhibits cytokine production by airway epithelial cells stimulated with diesel extract Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L33 - L39. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wan and D. Diaz-Sanchez Phase II Enzymes Induction Blocks the Enhanced IgE Production in B Cells by Diesel Exhaust Particles. J. Immunol., September 1, 2006; 177(5): 3477 - 3483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Mutlu, C. Snyder, A. Bellmeyer, H. Wang, K. Hawkins, S. Soberanes, L. C. Welch, A. J. Ghio, N. S. Chandel, D. Kamp, et al. Airborne Particulate Matter Inhibits Alveolar Fluid Reabsorption in Mice via Oxidant Generation Am. J. Respir. Cell Mol. Biol., June 1, 2006; 34(6): 670 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. W. Ryter, J. Alam, and A. M. K. Choi Heme Oxygenase-1/Carbon Monoxide: From Basic Science to Therapeutic Applications Physiol Rev, April 1, 2006; 86(2): 583 - 650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 J. Li, M. L. Spletter, and J. A. Johnson Dissecting tBHQ induced ARE-driven gene expression through long and short oligonucleotide arrays Physiol Genomics, March 21, 2005; 21(1): 43 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ohtani, S. Nakagawa, M. Kurosawa, M. Mizuashi, M. Ozawa, and S. Aiba Cellular Basis of the Role of Diesel Exhaust Particles in Inducing Th2-Dominant Response J. Immunol., February 15, 2005; 174(4): 2412 - 2419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. W. Cheng, W. W. Lee, C. H. Li, C. C. Lee, and J. J. Kang Genotoxicity of Motorcycle Exhaust Particles In Vivo and In Vitro Toxicol. Sci., September 1, 2004; 81(1): 103 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 T. P. J. Hofer, E. Bitterle, I. Beck-Speier, K. L. Maier, M. Frankenberger, J. Heyder, and L. Ziegler-Heitbrock Diesel exhaust particles increase LPS-stimulated COX-2 expression and PGE2 production in human monocytes J. Leukoc. Biol., May 1, 2004; 75(5): 856 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. D. Finkelman, M. Yang, T. Orekhova, E. Clyne, J. Bernstein, M. Whitekus, D. Diaz-Sanchez, and S. C. Morris Diesel Exhaust Particles Suppress In Vivo IFN-{gamma} Production by Inhibiting Cytokine Effects on NK and NKT Cells J. Immunol., March 15, 2004; 172(6): 3808 - 3813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
G. Kronke, V. N. Bochkov, J. Huber, F. Gruber, S. Bluml, A. Furnkranz, A. Kadl, B. R. Binder, and N. Leitinger Oxidized Phospholipids Induce Expression of Human Heme Oxygenase-1 Involving Activation of cAMP-responsive Element-binding Protein J. Biol. Chem., December 19, 2003; 278(51): 51006 - 51014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Xiao, M. Wang, N. Li, J. A. Loo, and A. E. Nel Use of Proteomics to Demonstrate a Hierarchical Oxidative Stress Response to Diesel Exhaust Particle Chemicals in a Macrophage Cell Line J. Biol. Chem., December 12, 2003; 278(50): 50781 - 50790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. Burdick, J. W. Davis II, K. J. Liu, L. G. Hudson, H. Shi, M. L. Monske, and S. W. Burchiel Benzo(a)pyrene Quinones Increase Cell Proliferation, Generate Reactive Oxygen Species, and Transactivate the Epidermal Growth Factor Receptor in Breast Epithelial Cells Cancer Res., November 15, 2003; 63(22): 7825 - 7833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Risom, M. Dybdahl, J. Bornholdt, U. Vogel, H. Wallin, P. Moller, and S. Loft Oxidative DNA damage and defence gene expression in the mouse lung after short-term exposure to diesel exhaust particles by inhalation Carcinogenesis, November 1, 2003; 24(11): 1847 - 1852. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Burstyn, P. Boffetta, D. Heederik, T. Partanen, H. Kromhout, O. Svane, S. Langard, R. Frentzel-Beyme, T. Kauppinen, I. Stucker, et al. Mortality from Obstructive Lung Diseases and Exposure to Polycyclic Aromatic Hydrocarbons among Asphalt Workers Am. J. Epidemiol., September 1, 2003; 158(5): 468 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
N. Hill-Kapturczak, C. Voakes, J. Garcia, G. Visner, H. S. Nick, and A. Agarwal A cis-Acting Region Regulates Oxidized Lipid-Mediated Induction of the Human Heme Oxygenase-1 Gene in Endothelial Cells Arterioscler Thromb Vasc Biol, August 1, 2003; 23(8): 1416 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Y. Chin, M. A. Trush, A. M. K. Choi, and T. H. Risby Transcriptional regulation of the HO-1 gene in cultured macrophages exposed to model airborne particulate matter Am J Physiol Lung Cell Mol Physiol, March 1, 2003; 284(3): L473 - L480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-L. Chen, S. E. Varner, A. S. Rao, J. Y. Grey, S. Thomas, C. K. Cook, M. A. Wasserman, R. M. Medford, A. K. Jaiswal, and C. Kunsch Laminar Flow Induction of Antioxidant Response Element-mediated Genes in Endothelial Cells. A NOVEL ANTI-INFLAMMATORY MECHANISM J. Biol. Chem., January 3, 2003; 278(2): 703 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Li, M. Wang, T. D. Oberley, J. M. Sempf, and A. E. Nel Comparison of the Pro-Oxidative and Proinflammatory Effects of Organic Diesel Exhaust Particle Chemicals in Bronchial Epithelial Cells and Macrophages J. Immunol., October 15, 2002; 169(8): 4531 - 4541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Rudra-Ganguly, S. T. Reddy, P. Korge, and H. R. Herschman Diesel Exhaust Particle Extracts and Associated Polycyclic Aromatic Hydrocarbons Inhibit Cox-2-dependent Prostaglandin Synthesis in Murine Macrophages and Fibroblasts J. Biol. Chem., October 11, 2002; 277(42): 39259 - 39265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li and J. A. Johnson Time-dependent changes in ARE-driven gene expression by use of a noise-filtering process for microarray data Physiol Genomics, June 3, 2002; 9(3): 137 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Koike, S. Hirano, N. Shimojo, and T. Kobayashi cDNA Microarray Analysis of Gene Expression in Rat Alveolar Macrophages in Response to Organic Extract of Diesel Exhaust Particles Toxicol. Sci., June 1, 2002; 67(2): 241 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Whitekus, N. Li, M. Zhang, M. Wang, M. A. Horwitz, S. K. Nelson, L. D. Horwitz, N. Brechun, D. Diaz-Sanchez, and A. E. Nel Thiol Antioxidants Inhibit the Adjuvant Effects of Aerosolized Diesel Exhaust Particles in a Murine Model for Ovalbumin Sensitization J. Immunol., March 1, 2002; 168(5): 2560 - 2567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Albini, M. Morini, F. D'Agostini, N. Ferrari, F. Campelli, G. Arena, D. M. Noonan, C. Pesce, and S. De Flora Inhibition of Angiogenesis-driven Kaposi's Sarcoma Tumor Growth in Nude Mice by Oral N-Acetylcysteine Cancer Res., November 1, 2001; 61(22): 8171 - 8178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Bonvallot, A. Baeza-Squiban, A. Baulig, S. Brulant, S. Boland, F. Muzeau, R. Barouki, and F. Marano Organic Compounds from Diesel Exhaust Particles Elicit a Proinflammatory Response in Human Airway Epithelial Cells and Induce Cytochrome p450 1A1 Expression Am. J. Respir. Cell Mol. Biol., October 1, 2001; 25(4): 515 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. De Flora, A. Izzotti, F. D'Agostini, and R. M. Balansky Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points Carcinogenesis, July 1, 2001; 22(7): 999 - 1013. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
44-luciferase reporter. The SX2-M2 mutant
contains base pair changes in the AP-1 site, leaving the ARE core
sequence intact. The SX2-M239 mutant contains base pair changes that
affect the ARE core sequence.
