1Laboratoire de Cytophysiologie et Toxicologie cellulaire, Université Paris 7 - Denis Diderot, 75251 Paris cedex 05; and 2Institut National de la Santé et de la Recherche Médicale U490, Centre universitaire des Saints-Pères, 75006 Paris, France
Submitted 6 December 2002 ; accepted in final form 30 April 2003
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ABSTRACT |
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cytochrome P-450 1A1; NADPH quinone oxidoreductase-1; antioxidant responsive element
In vitro toxicological studies have revealed that macrophages and
epithelial cells are the main effectors of this inflammatory response,
releasing proinflammatory cytokines involved in the recruitment of
inflammatory cells (3,
4,
11). In particular, we have
shown in previous studies that DEP induce the release of granulocyte
macrophage-colony stimulating factor (GMCSF) in nasal and bronchial epithelial
cells (5). This release results
from the phagocytosis of DEP and the activation of transduction pathways
(MAPK) and transcription factors like NF-B, which control transcription
of the GM-CSF gene (4,
5,
7). Comparison of the
respective roles of the carbonaceous core and organic compounds in the
molecular responses induced in respiratory epithelial cells has revealed that
organic compounds are important for GM-CSF release and MAPK and NF-
B
activation (6).
Two main families of organic compounds are adsorbed on DEP: PAH and quinones. PAH could be desorbed from DEP and become available to bind to the cytosolic aryl hydrocarbon receptor (AhR) and induce gene expression-like phase I metabolization enzymes, especially cytochrome P-450 1A1 (CYP1A1). The toxicity of PAH is known to be related to their bioactivation by CYP1A1. Indeed, PAH metabolism produces electrophilic and reactive metabolites, including reactive oxygen species (ROS). Some PAH metabolites can produce DNA adducts responsible for the genotoxicity of DEP if they are not detoxified by phase II metabolization enzymes.
Quinones are known to generate an oxidative stress by redox cycles. They are suspected to be responsible for the production of O2-· and ·OH radicals detected by electron paramagnetic resonance (EPR) in methanol extracts of DEP (23). The quinones could also be involved in indirect free radical production linked to the activity of enzymes like NADPH-cytochrome P-450 reductase (16). Indeed, this enzyme yields semiquinones that by autooxidation produce ROS. The detoxification of quinones occurs by two-electron reduction performed by NADPH-quinone oxidoreductase 1 (NQO-1).
We postulate that the production of ROS by DEP components could be a key
event triggering cellular activation leading to the inflammatory response.
Indeed, we have shown that antioxidants reduced both the induction of GM-CSF
release and the activation of NF-B by DEP or organic extracts of DEP
(OE-DEP) (6). Furthermore, Li
et al. (18) have shown that,
in macrophages, OE-DEP induced the expression of the antioxidant enzyme heme
oxygenase I (HO-1) via the antioxidant responsive element (ARE). Finally, we
have recently shown that DEP and OE-DEP induce CYP1A1 gene expression in
bronchial epithelial cells; this could lead to the metabolization of these
compounds and the release of ROS
(6).
The aim of this study was to contribute to a better understanding of the mechanism of action of DEP involving ROS in airway epithelial cells using two cellular models: primary cultures of human nasal turbinates or polyps (nasal cells) and the 16HBE14o-human bronchial epithelial cell line (16HBE cells) (9). First, we assessed ROS production in epithelial cells using fluorescent probes directly detecting either peroxide production or thiol depletion. We investigated the fate of DEP studying 1) the expression of the phase I metabolization enzyme gene CYP1A1 and CYP1A1 activity and 2) the expression of the phase II metabolization enzyme gene NQO-1 and its modulation by antioxidants. Furthermore, activation of the ARE was studied, as this cis-acting regulatory element is known to be activated by ROS and electrophilic compounds and is located in the 5'-flanking region of genes encoding antioxidant enzymes (HO-1) or metabolization enzymes (NQO-1). We present evidence that DEP, used at noncytotoxic concentrations, induced both dose-dependent peroxide production and thiol depletion that are mainly due to their organic component. Organic compounds, especially PAH, are likely to become available, since DEP induce CYP1A1 mRNA expression and increase CYP1A1 enzymatic activity. In addition, NQO-1 mRNA expression was induced by DEP and reduced in the presence of antioxidants. We also demonstrate, for the first time, an increased binding to the ARE of nuclear proteins, including Nrf2, in DEP-treated cells.
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MATERIALS AND METHODS |
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Cell culture and toxic treatment conditions. Dr. D. C. Gruenert (9) (Colchester, VT) kindly provided the human bronchial epithelial cell subclone 16HBE. The cell line was cultured in DMEM/F-12 culture medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (1%), fungizone (0.125 µg/ml, Invitrogen), and UltroserG (UG) (2%, Invitrogen). Cells were cultured on collagen (type I, 4 µg/cm2)-coated 25- or 75-cm2 flasks, 6- or 96-well plates (Costar, Cambridge, MA) at 20,000 cells/cm2. At the time of treatment, UG was not added to DMEM/F-12. Human nasal polyps or turbinates obtained from patients undergoing polypectomy or turbinectomy were cultured as previously described by Million et al. (21). Briefly, they were washed in DMEM/F-12 and incubated with 2 mg/ml of pronase (protease XIV) in DMEM/F-12 supplemented with 50 U-50 mg/ml of penicillin-streptomycin at 4°C for 16-20 h under rotary agitation (80 rpm). Ten percent fetal calf serum (FCS) was then added to neutralize the enzyme. After washing, the cell suspension was filtered on a 30-µm-diameter filter and centrifuged at 400 g for 5 min. The supernatant was eliminated, and cells were resuspended in 20 ml of DMEM/F-12. Aggregates were discarded, and dissociated cells were preplated for 2 h at 37°C on plastic dishes (Falcon Merck-Eurolab, Strasbourg, France) to eliminate most contaminating fibroblasts, and epithelial cells were counted. Cells were cultured on six-well plates at 500,000 cells/well for 6 days.
Cultures were incubated in humidified 95% air with 5% CO2 at 37°C. We made controls by using 0.04% DPL for DEP and CB or 0.1% DMSO for OE-DEP and PAH. The different antioxidants tested on the 16HBE cells were added in the culture medium 30 min before treatment with DEP or OE-DEP.
Analysis of intracellular ROS levels. Intracellular ROS levels were assessed with H2DCF-DA, an oxidation-sensitive fluorescent probe. Once inside the cell, this probe is deacetylated by intracellular esterases forming H2DCF, which in the presence of a variety of intracellular peroxides is oxidized to a highly fluorescent compound, 2',7'-dichlorofluorescein (DCF). Stock H2DCF-DA solution was made at 20 mM in DMSO and stored at -20°C. Before the toxic treatment, cells were loaded for 20 min with 20 µM H2DCF-DA in Hanks' balanced salt solution (HBSS) either for microplate or cytometric fluorescent analysis. Positive control was obtained with t-BHP, and we verified that the toxins do not directly oxidize the probe. All results are expressed in relative units normalized to the control.
Analysis of intracellular thiol levels. Cellular thiol levels were analyzed with mBBr, which forms a fluorescent adduct with sulfhydryl groups. mBBr was made up as a 4 mM solution in 100% ethanol and stored at -20°C. After the toxic treatment, cells were labeled with 40 µM mBBr for 10 min at room temperature in HBSS (Invitrogen) in the dark according to Hedley and Chow (13). After labeling and rinsing, we quantified the fluorescence either with the fluorescent plate reader (Fluostar BMG Labtechnologies, Champigny-sur-Marne, France) using an excitation wavelength of 480 nm and an emission wavelength of 538 nm or with a flow cytometer. In the last case, cells were detached by trypsination, and trypsin action was stopped by addition of 10% FCS (Invitrogen). Cells in suspension were then kept on ice until flow cytometric analysis. Treatment with 0.1 mM NEM for 2 min before mBBr labeling was done as a positive control. All results are expressed in relative units normalized to the control.
DCF and mBBr fluorescence analysis by FACS. Before the fluorescence analysis performed with an EPICS-Elite-ESP flow cytometer (Coultronics-France), cells were labeled with 3 µg of propidium iodide (PI) per milliliter to ascertain viability. A 15-mW air-cooled argon-ion laser tuned at 488 nm was used for DCF fluorescence, and an Innova 90-5 argon-ion laser (Coherent-France) running at 100 mW of output in multilane 351-363 nm mode was used for mBBr fluorescence. DCF, mBBr, and PI fluorescence were collected, respectively, though a 525-, 470-, and 620-nm band-pass filter. Forward-angle light scatter and right-angle scatter was used to select cells.
RNA isolation and Northern blot analysis. Total cellular RNA was isolated from subconfluent cells (10 x 106) cultured in 75-cm2 flasks, by using the Tri reagent according to the manufacturer's instructions. The amount of RNA in aqueous solution was determined by absorbance at 260 nm. Equal amounts (30 µg) of total cellular RNA were separated by size by 0.65 M formaldehyde-agarose (0.8%) gel electrophoresis and transferred onto Hybond-Plus membranes (Amersham Biosciences, Orsay, France) using 10x SSC buffer (1x: 150 mM NaCl, 15 mM sodium citrate). The blot was then baked for 2 h at 80°C and hybridized to specific probes. The blots were prehybridized at 65°C in rapid Hybrid solution (Amersham) and hybridized overnight at 65°C with 1-5 x 107 cpm/ml of 32P-labeled cDNAs. Probes were synthesized from cDNAs with the Megaprime DNA labeling kit (Amersham) according to the manufacturer's instructions. After hybridization, the blots were washed for 30 min at 65°C with 2x SSC, then 1x SSC, then 0.5x SSC, in the presence of 0.1% SDS. The membranes were exposed, and the radioactivity was quantified with PhosphoImager and ImageQuant software (Molecular Dynamics).
Dr. I. De Waziers and Dr. R. Barouki provided the cDNA probes of the CYP1A1 and NQO-1 genes, respectively.
Semiquantitative reverse transcriptase-polymerase chain reaction. We reverse-transcribed 1 µg of total RNA into cDNA using Oligo-dT Primer and Superscript II RNase H- Reverse Transcriptase (Invitrogen).
For semiquantitative experiments, an aliquot of cDNA libraries was amplified by 23 cycles of PCR with NQO-1 oligonucleotides or with GAPDH oligonucleotides in a PTC-100 thermocycler (MJ Research, Watertown, MA). Amplified products were then separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
Evaluation of CYP1A1 activity by ethoxyresorfin-O-deethylase test. Subconfluent primary nasal cells were treated with 10 µg/cm2 DEP or 3 µM B(a)p for 24 h. After being washed with phosphate-buffered saline (PBS, Invitrogen), cells were incubated with DMEM/F-12 containing 5 µM ethoxyresorufin and 2 mM salicylamide. Ethoxyresorufin is metabolized by CYP1A1 in resorufin, which is a fluorescent compound. Kinetic fluorescence measurements were made with a microspectrofluorimeter (Fluostar Galaxy, BMG Labtechnologies) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm for 40 min.
EMSA. After treatment, nuclear extracts were isolated from subconfluent 16HBE cells (3 x 106) cultured in 25-cm2 flasks as described by Staal et al. (25). Cells were washed and removed by scraping in Tris-buffered saline (25 mM Tris · HCl, 136 mM NaCl, 2.7 mM KCl, pH 7.4) and pelleted. The pellets were resuspended in 400 µl of ice-cold hypoosmotic buffer (10 mM HEPES, 10 mM KCl, 2 mM MgCl2, and 0.1 mM EDTA, pH 7.8) supplemented with 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml antipain, 0.3 µg/ml leupeptin, and 0.5 µg/ml pepstatin. Nuclei were spun down at 16,000 g for 30 s after addition of a 10% Nonidet P-40 solution and then resuspended in 40 µl of a hyperosmotic buffer (50 mM HEPES, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, and 10% glycerol, pH 7.8) supplemented with 0.5 mM DTT, 0.5 mM PMSF, 1 µg/ml antipain, 0.3 µg/ml leupeptin, and 0.5 µg/ml pepstatin. Nuclear proteins were extracted by incubating the nuclei for 30 min at 4°C, with a slow rotation followed by a 16,000-g centrifugation for 10 min. The supernatants containing the nuclear extracts were stored until use at -80°C. They were complexed with different radiolabeled double-stranded oligonucleotides: 1) human consensus ARE containing the human (h) ARE core sequence of glutathione-S-transferase (GST) or NQO-1 shown in bold letters (Fig. 6) (synthesized by Invitrogen), 2) different mutant hARE with mutations in the hARE core sequence of NQO-1 (hARE mutant 1, hARE mutant 2, hARE mutant 3, Invitrogen; Fig. 7) and activator protein (AP)-1 consensus (Invitrogen) oligonucleotides. For cold competition, 100-fold excess unlabeled probe (hARE, AP-1) was incubated for 15 min before addition of the labeled probe. Shifted complexes were electrophoresed on 5% polyacrylamide gel as described by Baeza-Squiban et al. (2).
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Immunocytochemistry. 16HBE cells were grown on slides in DMEM/F-12 medium with or without 10 µg/cm2 DEP, 10 µg/ml OE-DEP or 3 µM B(a)p for 4 h. The cells were then washed with PBS (Invitrogen) supplemented with 0.1% Tween 20 and 3% BSA. The cells were fixed in paraformaldehyde at room temperature for 20 min. After being washed with PBS/Tween 20/BSA, the cells were incubated with rabbit anti-human Nrf2 antibody (100-fold dilution; Santa Cruz Biotechnology) in PBS/Tween 20/BSA overnight at 4°C, and after washing were incubated with FITC-conjugated goat anti-rabbit immunoglobulin antibody (200-fold dilution, Zymed) for 1 h at room temperature. After a last washing with PBS/Tween 20, a 4,6-diamidino-2-phenylindole solution at 0.5 µg/ml in PBS/Tween 20 was placed on the slides for 2 min. The cells were then examined by confocal fluorescence microscopy.
Statistical analysis. All data were expressed as the means
± SE of three cultures (for six-well plates) or of eight cultures (for
96-well plates) from a representative experiment. Means were compared by
analysis of variance. The equal variance test is significant with =
0.05 (P < 0.001). All pairwise multiple comparisons were made with
the Student-Newman-Keuls method (t-test, P < 0.05).
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RESULTS |
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Because PAH are the main organic compounds of DEP, several of them were tested at their relevant concentration (i.e., corresponding to their amount in DEP): phenanthrene (20 nM), fluoranthene (10 nM), chrysene (3 nM), pyrene (10 nM), B(a)p (0.5 nM), and 1-nitropyrene (4 nM). Only phenanthrene and 1-nitropyrene exhibited a statistically significant increase of DCF fluorescence in 16HBE cells (Table 1). The OEDEP-induced increase of DCF fluorescence appears less important when evaluated by microspectrofluorimetry (Table 1) in comparison with flow cytometry (Fig. 1). Indeed, DCF fluorescence was evaluated in the whole culture well with viable and dead cells by microspectrofluorimetry, whereas flow cytometry was used to measure DCF fluorescence cell-by-cell in viable cells (determined by PI incorporation).
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The antioxidants NAC (10 mM) and mannitol (10 mM) have no significant effect on the basal DCF fluorescence intensity of 16HBE cells, but they reduce the DCF fluorescence intensity induced by 10 µg/ml of OE-DEP, in particular NAC (Fig. 2A). The same results were obtained with the antioxidant enzyme catalase at 1,400 U/ml (Fig. 2B).
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The mBBr probe becomes fluorescent when alkylated by thiols and allowed us to determine the intracellular thiol levels. In 16HBE cells, both DEP and OE-DEP provoke a dose-dependent depletion of intracellular thiols that is smaller than that induced by NEM (0.1 mM), which is known to alkylate all intracellular thiols (Table 2).
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DEP and OE-DEP induce CYP1A1 and NQO-1 mRNA expression. The expression of metabolization enzymes was studied by Northern blot experiments of total RNA from 16HBE cells treated with control buffer or DEP (10 µg/cm2), OE-DEP (15 µg/ml), CB (10 µg/cm2), or B(a)p (3 µM), a PAH known to induce the expression of CYP1A1 and NQO-1 genes. A typical time-course study shown in Fig. 3A revealed that CYP1A1 is not expressed in control cultures. CYP1A1 expression appears following 2 h of treatment with DEP or OE-DEP (Fig. 3A). Its expression was clearly increased and maximal at 6 h, then it decreased at 24 h and returned to basal levels after 48 h. This time course is similar to that obtained when B(a)p was used as a positive control. In contrast, CB did not induce CYP1A1 mRNA expression. A similar study on primary culture of human nasal cells (Fig. 3B) treated for 6 h with DEP or OE-DEP confirmed the clear induction of CYP1A1 mRNA by these compounds.
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The Northern blots were reprobed with a 32P-labeled cDNA for NQO-1 mRNA (Fig. 3, C and D). Two transcripts were observed that were already present in control cultures. However, their expression increased following 6 h of treatment with DEP, OE-DEP, or B(a)p; remained relatively high at 24 h; and returned nearly to basal levels at 48 h. Similar to CYP1A1, CB had no effect on NQO-1 mRNA expression. The induction of NQO-1 mRNA after 6 h of exposure was also observed in primary cultures of human nasal cells treated with DEP or OE-DEP (Fig. 3D).
By RT-PCR, the increase of NQO-1 mRNA expression induced by DEP was also observed after 24 h of treatment. When cells were cotreated with DEP and the antioxidants (NAC, catalase) (Fig. 4), NQO-1 mRNA expression was reduced and similar to the level of expression in their respective controls.
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DEP increases CYP1A1 enzymatic activity. We have shown that DEP induces CYP1A1 mRNA expression. We therefore confirmed CYP1A1 enzymatic activity using the ethoxyresorufin-O-deethylase (EROD) assay. In control nasal cells, no increase of fluorescence was detected for 40 min, but when cells were treated with DEP (10 µg/cm2) or B(a)p (3 µM), a time-dependent linear increase in fluorescence was observed (Fig. 5).
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DEP and OE-DEP increase protein binding to ARE. Because DEP and OE-DEP increase ROS production, the binding of proteins to the ARE was evaluated by EMSA. The ARE sequence of the human GST promoter hARE (GST) was first used; it is known to bind predominantly Nrf2, a transcription factor sensitive to oxidative stress. As shown in Fig. 6A, two protein-ARE complexes (C1 and C2) were detected with nuclear protein extracts of 16HBE cells treated with DEP at 30 µg/cm2. These two complexes are specific, since they are displaced by an excess of unlabeled hARE (GST) oligonucleotide. The most shifted complex, C2, is displaced by an excess of AP-1 oligonucleotides, suggesting that Jun or Fos could be involved in this complex. In contrast, the C1 complex is not modified by the AP-1 competitor oligonucleotide and is displaced by unlabeled hARE (GST).
Because NQO-1 expression is induced by DEP treatment and the NQO-1 promoter contains an ARE-like element, gel shift experiments using the ARE of the NQO-1 promoter, hARE (NQO-1), were performed. We also detected several complexes (C1' and C2') with the hARE (NQO1) probe (Fig. 6B). The C1' complex was further characterized using different mutated hARE (NQO-1) probes (Fig. 6). Compared with the wild-type hARE (NQO-1) sequence, mutant 1 displays a modified TGAC sequence, which is required both for a functional ARE and for binding of AP-1. In the case of mutant 2, the AP-1-specific TCA sequence is altered, whereas mutant 3 displays an altered ARE-specific GC site. The binding profiles of the B(a)p-treated 16HBE nuclear extracts for the wild-type labeled hARE (NQO-1) and mutant labeled hARE (NQO-1) oligonucleotides are shown in Fig. 6B. Striking differences in the intensity of the complexes are observed between the different oligonucleotides. The migration and abundance of the C1' complex were similar in the case of the wild-type and the mutant 2 sequence, whereas the abundance of complex C1' was lower in the case of mutant 3. Interestingly, no complexes were formed using the mutant 1 probe. Together, these data suggest that although the other complexes are related to the AP-1 complex, the C1' complex is more specific for the ARE sequence. We studied the effect of DEP and OE-DEP on the abundance of this complex. As shown in Fig. 7, both treatments lead to an increase in C1 and C1' complex formation with either the GST or the NQO-1 probes. C1 and C1' are probably similar to the ARE-specific complexes and different from AP-1 complexes. Both are increased in DEP-treated cells.
OE-DEP induces nuclear translocation of the transcription factor Nrf2. To determine whether Nrf2 is regulated by DEP, we studied nuclear translocation of Nrf2. The transcription factor Nrf2 was immunodetected in 16HBE cells treated with either control buffer or 10 µg/cm2 DEP, 10 µg/ml OE-DEP, or 3 µM B(a)p for 4 h. Contrast-phase microscopy of the same cells verified the localization and the integrity of the nuclei. In control cultures, Nrf2 was predominantly found in the cytoplasm (Fig. 8). When the cells were treated with B(a)p (positive control), a clear nuclear translocation of Nrf2 was observed (Fig. 8). Cells treated with DEP or OE-DEP exhibited an intermediate situation: Nrf2 was translocated in the nuclei but to a lesser extent than with B(a)p.
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DISCUSSION |
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ROS production was evaluated in human airway epithelial cells exposed to native DEP, OE-DEP, or CB, a model of carbonaceous core, to determine which component is mainly responsible for this production. It clearly appears that only DEP and their organic extracts induced a dose-dependent modification of the redox state of human airway epithelial cells. Previous studies in macrophages show that high concentrations of OE-DEP (100 µg/ml) triggered peroxide production (14, 22). In contrast, the carbonaceous core of 95 nm diameter mimicked by CB did not induce peroxide production, even at high concentrations.
Several additional observations suggest that the organic compounds are most likely the main contributors of ROS production: 1) OE-DEP used at 10 µg/ml produced a similar amount of peroxides as DEP at 10 µg/cm2; 2) some PAHs, known to be present in DEP and used at their real concentration in DEP, can trigger peroxide production that was only significant for phenanthrene and 1-nitropyrene; and 3) cotreatment with antioxidants reduced OE-DEP-induced peroxide production. Different antioxidants were used to determine which ROS were produced and where they were produced. A partial reduction occurs with the extracellular antioxidant mannitol, suggesting that ROS could be generated outside the cells. This is consistent with the demonstration of O2-· and ·OH radical production by a methanol extract of DEP measured by EPR (16, 23) involving quinones. Quinoid redox cycling is also proposed as a mechanism for sustained free radical generation by airborne particulate matter with an aerodynamical diameter <2.5 µm (PM2.5) (24). The proposal is further strengthened by the results obtained with the antioxidant enzyme catalase, which may scavenge extracellular, DEP-produced H2O2. The antioxidant NAC, known to enter cells, inhibits OEDEP-induced peroxide production, suggesting that ROS can enter cells or could be produced in cells. An integrated question is the mechanism by which DEP provoke an oxidative stress within the cells.
We hypothesized that intracellular ROS production could be partially due to PAH metabolism, which requires the CYP1A1 enzyme. Indeed the CYP1A1 catalytic activity is postulated to generate ROS (unpublished data) as well as reactive metabolites such as PAH-O-quinones, which could produce ROS by redox cycles. Evidence for PAH metabolization was indirect and relied on investigating CYP1A1 gene expression, which is specifically induced by PAH. We observed in previous preliminary experiments a clear CYP1A1 mRNA induction in DEP- or OE-DEP-treated 16-HBE cells (6). We have now confirmed this observation on primary cultures of human nasal cells, and we have characterized its time course. Induction starts at 2 h of treatment but is transient and is almost back to basal levels at 24 h. The kinetics are nearly the same for DEP as for OE-DEP and B(a)p, suggesting that organic compounds could be quickly desorbed from DEP. Indeed this desorption is a prerequisite for PAH to interact with the AhR and activate the CYP1A1 gene, leading to an increase in CYP1A1 enzymatic activity. In addition to CYP1A1 mRNA induction, both DEP- and OE-DEP-treated cells exhibited increased expression of NQO-1 mRNA, occurring later than that of CYP1A1. The delayed induction of NQO-1 argues in favor of an indirect mechanism. Indeed, antioxidants partially reduce DEP-induced NQO-1 expression, suggesting that NQO-1 is induced by ROS, which could result either from the induction of CYP1A1 by DEP organic compounds or from the redox cycling of quinones. Thus DEP organic compounds could influence gene expression sequentially, first through AhR activation and then through generation of ROS by phase I metabolizing enzymes.
Together, these data suggest that DEP organic compounds are bioavailable and are probably metabolized, since CYP1A1 enzymatic activity is increased, as we first showed by an EROD test. They support the in vivo observation of increased CYP1A1 activity and protein levels in pulmonary microsomes from rats (19) or mice (1) exposed to DEP. In addition, they shed new insight on experiments on dogs exposed to aerosol boluses of B(a)p adsorbed on denuded diesel soot, showing that 20% of the released PAH are deposited on the tracheobronchial epithelium and are adsorbed and metabolized, whereas 80% are deposited in the alveolar region, where they rapidly passed into the blood without such metabolism (12).
As mentioned above, the expression of NQO-1, one of several phase II metabolization and antioxidant enzymes, is under the control of the ARE. This genetic response element is known to be activated by ROS and electrophilic compounds. The demonstration of its activation provides additional evidence for the biological impact of ROS production and DEP organic compound metabolization in DEP-treated 16HBE cells. By mobility shift assay, we demonstrate for the first time that in human bronchial epithelial cells, DEP and their extracts dose dependently increased the formation of a complex between an ARE consensus sequence and nuclear proteins, suggesting that ARE activation could occur and be responsible for the induction of NQO-1 gene expression. Such activation has already been shown in macrophages exposed to 3,6-benzo(a)pyrene quinone, an oxidation metabolite of B(a)p, which could explain the induction of the antioxidant enzyme HO-1 by the polar fraction of OE-DEP (18).
Recent evidence suggests that Nrf2 is a critical transcription factor for the regulation of ARE-containing genes that encode antioxidant proteins. These genes include phase II genes and other genes such as the HO-1 gene (15). Thus the critical role of Nrf2 has been supported by knockout mice. Indeed nrf2(-/-) mice exhibit significant reduction of constitutive and/or inducible phase II enzymes (8). Recently, it was shown that both oxidative and PAH-derived DNA adducts formation are accelerated in the lungs of nrf2(-/-) mice compared with nrf2(+/-) mice upon exposure to DEP, whereas the CYP1A1 mRNA levels are similar in both types of mice (1). Our in vitro investigations indicate that in DEP-treated 16HBE cells, Nrf2 is likely involved in the ARE binding proteins, since nuclear translocation of Nrf2 was observed in treated cells.
In conclusion, we have shown that in airway epithelial cells, DEP, via
their organic components, modify the cellular redox state. We provide evidence
that organic compounds are bioavailable as they induce phase I (CYP1A1) and
phase II (NQO-1) gene expression and can be metabolized, as CYP1A1 enzymatic
activity is increased. Resulting ROS and reactive metabolites likely
contribute to the increased binding of proteins such as Nrf2 to an hARE
consensus sequence involved in the expression of the NQO-1 gene. Finally DEP
and their extracts induce, in human bronchial and nasal epithelial cells, the
expression of numerous genes implicated in detoxification that are activated
via xenobiotic responsive element and ARE as well as in the secretion of
proinflammatory cytokines via NF-B-responsive element
(6).
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DISCLOSURES |
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This work was also supported by Caisse d'Assurance Maladie des Professions Libérales de Province, Paris, France.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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