Introduction

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Suspended particulate matter with diameters less than 2.5 µm (PM2.5) have been considered to be important in inducing airway inflammatory responses because they precipitate easily in small airways (diameters less than 2 mm) for a long period of time to elicit disease (1). Diesel exhaust particles (DEPs), the major component of PM2.5 in urban areas, have been demonstrated to stimulate cytokine production in vitro and in vivo (4). Salvi and associates (8) found that acute exposure to diesel exhaust induced airway inflammatory responses with cell infiltration and increased mediator production in healthy human volunteers. It has been demonstrated that DEPs stimulated human airway epithelial cells to release cytokines and mediators such as interleukin (IL)-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), and the soluble form of intercellular adhesion molecule-1 in vitro (7, 9). Most of these studies used DEPs as suspended forms, and a variety of organic compounds including hydrocarbons may be eluted from DEPs and induce such biologic effects on the cells. However, it remains unknown what component(s) derived from DEPs are important for their activities.

In the present study we investigated the effects of DEP-derived components on IL-8 gene expression and nuclear factor (NF)-B activation in human airway epithelial cells in vitro. We also compared the activity of each component to induce release of cytokines/chemokines, including GM-CSF and regulated on activation, normal T cells expressed and secreted (RANTES) as well as IL-8. The studies showed that DEP-induced IL-8 expression and NF-B activation were replicated only by benzene-derived components in a human bronchial epithelial cell line, BEAS-2B, and in human airway epithelial cells obtained from very peripheral airways by an ultrathin bronchoscope. Finally, benzo[a]pyrene (BaP), an important component mainly contained in benzene-extracted parts, also showed such biologic activity in vitro.

	Materials and Methods

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Preparation of DEPs and Their Components

The engine used for preparation of DEPs was a 4JB1 type (Isuzu Automobile Co., Tokyo, Japan), light-duty (2740 cc), four-cylinder diesel engine. The engine was connected to an EDYC dynamometer (Meiden-Sya, Tokyo, Japan), and was operated using a standard diesel fuel with speeds of 1,500 rpm under the load of 10 torque (kg/m). The exhaust was introduced into a stainless-steel dilution tunnel (300 mm  × 8,400 mm). The DEPs were collected on a glass fiber filter (203 × 254 mm) in a constant-volume sampler system equipped to the end of the dilution tunnel. The temperature at the sampling point was below 50°C. The diameter of the particles was measured by an Anderson Air Sampler of the low-pressure type (12), and the mean diameter was 0.4 µm. Most of the shapes analyzed by a scanning electron microscope were globular. The details on the DEPs we used were previously described (4, 7).

For characterization of the components extracted from DEPs, DEPs (2 g) suspended in 100 ml of n-hexane were sonicated for 10 min under cooling conditions by an Ultrasonic Disruptor UD-201. The suspension was filtered and the residue was treated with the same volume of benzene, followed successively by chloroform, ethyl acetate, and methanol. The order of solvents used for extraction procedure was dependent on the polarity of each solvent, namely from more lipid-soluble to less lipid-soluble solvent, followed by the protocol previously reported (13). The amounts recovered in each extract were 0.66 g (n-hexane), 0.22 g (benzene), 0.18 g (chloroform), 0.11 g (ethyl acetate), and 0.20 g (methanol). Each fraction was evaporated and then dissolved in methanol/acetone (1:1, vol/vol) to make a final concentration of 10 mg/ml as stock. Preliminary experiments showed that diluted methanol/acetone (vehicle alone) showed no significant cytotoxicity and no effect on IL-8 messenger RNA (mRNA) levels or on cytokine/chemokine release in the ranges of the present experiments.

BaP was obtained from Sigma, RBI (St. Louis, MO) and was dissolved in dimethyl sulfoxide (DMSO). The stock was further diluted in culture media to the final concentrations, keeping the final carrier concentration less than 0.1%. At these concentrations, DMSO showed no effect on cytokine gene expression or release (data not shown).

Culture of Bronchial Epithelial Cell Line BEAS-2B

Human bronchial epithelial cell line BEAS-2B (14) (a kind gift from Drs. J. F. Lechner and C. C. Harris, National Cancer Institute, Bethesda, MD) was plated onto collagen-coated, 24-well, flat-bottomed tissue culture plates (Koken, Tokyo, Japan) at the density of 5 × 10⁴ cells/well in hormonally defined Ham's F12 medium (HD-F12) as reported (7, 15). HD-F12 contained 1% penicillin-streptomycin, 5 µg/ml insulin (GIBCO BRL, Grand Island, NY), 5 µg/ml transferrin (GIBCO), 25 ng/ml epidermal growth factor (Collaborative Research Corp., Lexington, MA), 15 µg/ml endothelial cell growth supplement (Collaborative Research), 2 × 10¹⁰ M triiodothyronin (GIBCO), and 10⁷ M hydrocortisone (GIBCO). The cells were incubated in a humidified atomsphere at 37°C and 5% CO₂. The medium was changed at Day 1 and subsequently every 2 d. Confluent monolayers of epithelial cells were stained with antikeratin (KL-1; Immunotech, Marseille, France) or antivimentin (DAKO-Vimentin; DAKOPatts, Glostrup, Denmark), or with control immunoglobulin (Ig) G1 monoclonal antibodies using avidin-biotin complex method.

Isolation and Culture of Very Peripheral Airway Epithelial Cells

To assess the effects of DEPs and their components on cytokine/ chemokine production by normal human peripheral airway epithelium, where DEPs may precipitate for a long period, bronchial epithelial cells were obtained from 10 healthy volunteers (all males, mean age 54.8 yr; all non- or ex-smokers) under fiberoptic bronchoscopy as previously reported (15, 16). They were free of any respiratory symptoms for the past 3 mo, with no abnormal chest X-ray findings, and received no treatment. The study was planned according to the ethical guidelines following the declaration of Helsinki and received institutional approval. An informed consent was obtained from each subject. Briefly, under fluoroscopic guidance an ultrathin fiberscope (BF-2.7T, outer diameter 2.7 mm with a biopsy channel of 0.8 mm; Olympus, Tokyo, Japan) was inserted through a 2.8-mm-diameter biopsy channel of BF-20. A BC-0.7T brush was then inserted to collect cells by brushing the airway mucosal surfaces under direct vision. The epithelial cells were harvested by vortexing the brush in media containing 10% fetal calf serum. The number of cells was counted by a standard hemocytometer and the cell viability was assessed by trypan blue dye exclusion technique. The cells were routinely stained by antikeratin antibody by a method described earlier, and only those samples with more than 95% positive staining were used in the study. The cell number was 1.48 ± 0.77 × 10⁶ and the viability was 71.2 ± 9.9% for the cells from very peripheral airways. The cells were cultured at the density of 1 × 10⁵/ml in commercially available SBGM media (Wako Junyaku, Tokyo, Japan). Fourth- and fifth-passaged cells were used in the experiments.

Northern Blot Analysis for IL-8 mRNA Expression in Human Bronchial Epithelial Cells

Northern blot analysis was performed to study the effects of DEPs and their components on IL-8 mRNA expression in human bronchial epithelial cells by the method described previously (15). Briefly, total cellular RNA was extracted by the method of Chomczynski and Sacchi (17) and electrophoresed on formaldehyde-denatured agarose gel (10 µg/lane), followed by capillary transfer onto a Biodyne nylon membrane (15). RNA integrity and equivalency of loading were routinely evaluated by ethidium bromide fluorescence. Blots were baked, prehybridized, and hybridized with 32P 5' end-labeled oligonucleotide probes specific for human IL-8 and -actin. The probes used in this study were reported previously (15). Blots were stringently washed after hybridization and exposed to X-ray film.

Electrophoretic Mobility Shift Assay

After the cells were washed with phosphate-buffered saline, the nuclear proteins were isolated by the method reported previously (18), with modifications. In brief, 2 to 3 × 10⁶ cells were harvested with the addition of trypsin-ethylenediamenetetraacetic acid (EDTA) solution (GIBCO), rinsed in Tris-buffered saline, resuspended in lysis buffer (10 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid [Hepes], 10 mM KCl, 0.1 mM N-2-hydroxyethylpoperazine-N'-ethane sulfonic acid [EGTA], 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]), and incubated on ice for 15 min. Nonidet P-40 (10%) was added to lyse the cells, and then the cells were centrifugated for 6 min at 4°C at 600 × g. The nuclear pellet was resuspended in extraction buffer (20 mM Hepes, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) and vortexed for 15 min on ice. The nuclear extract was centrifuged for 15 min at 12,000 rpm at 4°C. The supernatant was collected, divided into aliquots, and stored at 70°C. Protein concentration was determined by the Bradford dye-binding procedure (Bio-Rad Protein Assay), standardized with bovine serum albumin.

For the detection of the NF-B DNA binding, a NF-B-binding protein detection kit (GIBCO) was used. The sequences of the oligonucleotides containing a tandem repeat of the consensus sequence for the NF-B DNA binding site (underlined) were:

5'-GATCCAAGGGGACTTTCCATGGATCCAAGGGGACTTTCCCATG-3'

and 3'-GTTCCCCTGAAAGGTACCTAGGTTCCCCTGAAAGGTACCTAG-5'. Synthetic double-stranded oligonucleotides were labeled with -³²P-adenosine triphosphate using T4 polynucleotide kinase as recommended by the manufacturer.

The DNA binding reaction was conducted at room temperature for 20 min in a volume of 25 µl. The reaction mixture contained 10 µg nuclear extract, 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 4% (vol/vol) glycerol, 0.08 mg/ml sonicated salmon sperm DNA, and ³²P-labeled double-stranded oligonucleotides at 0.7 fmol/µg nuclear extract. After incubation, the samples were loaded onto a 4% polyacrylamide gel (polyacrylamide/bis (30%:0.8% wt/vol), 2.5% glycerol in 0.5 × Tris-borate-EDTA) and run at 120 V for 2 h. Each gel was then dried and subjected to autoradiography (18).

For supershift studies, 2 µl of anti-p65, anti-p50, or control antisera were added to the reaction mixture containing the B oligonucleotide. Binding of the antibody to the appropriate transcriptional factor was indicated by a supershift in the electrophoretic mobility shift assay (EMSA).

Cytokine Assay

Specific immunoreactivity for IL-8, GM-CSF, and RANTES in culture supernatants was measured by enzyme-linked immunosorbent assay kits (R&D Systems, Inc., Minneapolis, MN). Each sample was assayed in duplicate as recommended by the manufacturer.

Effects of the Drugs that Inhibit Signal Transduction

To study the mechanisms of IL-8 gene expression by each DEP component, various concentrations of the protein kinase C inhibitor staurosporin, antioxidant drugs with inhibitory effect on NF-B activation pyrrolidine dithiocarbamate (PTDC) (Sigma) and N-acetyl cysteine (NAC) (Sigma), and a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (a kind gift of SmithKline Beecham, Tokyo, Japan) (19) were added to the cells 1 h before the stimulation with DEPs or one of their components, and the effects of the drugs on IL-8 mRNA and IL-8 protein production were studied.

Statistical Analysis

The results were analyzed by Student's t test for comparison between the two groups, and by nonparametric equivalents of analysis of variance (ANOVA) for multiple comparison as reported (8).

	Results

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Cytotoxicity due to DEP Components

Cytotoxicity of DEPs and each of their extracts was evaluated by trypan blue dye exclusion technique, lactate dehydrogenase release assay, and a colorimetric 3-4,5-(dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium assay (Boehringer Mannheim, Mannheim, Germany). These studies demonstrated that DEPs and their extracts were not toxic to epithelial cells as compared with baseline (vehicle alone) data when evaluated after 24 and 48 h at no more than 50 µg/ml and their comparable concentrations (data not shown).

Effects of DEPs and Their Components on IL-8 mRNA Levels in Human Bronchial Epithelial Cells

To evaluate which component is a major part of the activity, we analyzed the activity of chemical components in DEPs. As shown in Figure 1, each of the extracted chemical components from DEPs including n-hexane extracts, benzene extracts, chloroform extracts, ethyl acetate extracts, and methanol extracts, was added to the cells (at the concentration equivalent to 25 µg/ml of original DEPs for 12 h).

View larger version (44K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Figure 1.   Effects of DEPs and their components on IL-8 mRNA levels in human bronchial epithelial BEAS-2B cells in vitro. BEAS-2B cells were cultured in hormonally defined HD-F12 until confluence. The cells were then treated with DEPs or each component. The cellular RNA was extracted after 12 h incubation and Northern blot analysis was performed to evaluate the changes in IL-8 mRNA levels. Benzene extracts as well as DEPs induced a significant increase in IL-8 mRNA levels, whereas the other components showed no such effects. (a) A representative result at 25 µg/ml of DEPs and equivalent dose of each component. (b and c) Results from three separate experiments are shown at (b) 5 and (c) at 25 µg/ml (*P < 0.01, ANOVA). (), vehicle alone = 100%. Data are presented as means ± standard error of the mean (SEM).

DEPs showed a stimulating effect on IL-8 gene expression, as reported previously (18). Among the five components, benzene-extracted components showed a significant stimulatory effect on IL-8 mRNA expression when studied at the dose equivalent to 5 and 25 µg/ml of original DEPs (Figure 1). As shown in Figures 2a and 2b, the benzene-extracted component showed a time- and dose-dependent stimulatory effect on IL-8 mRNA expression. In contrast, the addition of other components showed no significant upregulation of IL-8 mRNA levels (Figure 1).

View larger version (31K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   Figure 2.   Time- and dose-dependent effect of benzene extracts on IL-8 mRNA expression. (a) Results showing time dependency by Northern blot analysis. Upper panel: a representative result; lower panel: results from three separate experiments for time dependency (equivalent dose of 25 µg/ml of original DEPs) (*P < 0.01, ANOVA). (b) Results showing dose dependency evaluated after 12 h. Upper panel: a representative result; lower panel: results from three separate experiments for dose dependency (*P < 0.01, ANOVA). Data are presented as means ± SEM. (c) Effects of components on small airway epithelium. A representative result with normal human small airway epithelial cells is shown.

We further evaluated the effect of DEP components on small airway epithelial cells. As shown in Figure 2c, there was a significant increase in IL-8 mRNA levels in human normal peripheral epithelium when stimulated with benzene-extracted components for 12 h.

Effects of the Drugs on IL-8 mRNA Expression Induced by DEPs and Their Components

DEP-induced IL-8 gene expression was significantly attenuated by the pretreatment with PDTC, NAC and SB203580 in a dose-dependent fashion, but not with staurosporin (Figure 3a). These results suggest that oxidants-mediated and p38MAPK-mediated pathways might be involved in DEP-induced upregulation of IL-8 mRNA levels. When these three compoundsPDTC, NAC, and SB203580were pretreated, the stimulatory effect of benzene-extracted components on IL-8 mRNA levels was almost completely abolished, as shown in Figure 3b, suggesting that the activity of this component was also p38MAPK- and oxidants-mediated.

View larger version (34K): [in this window] [in a new window]   Figure 3.   Effects of drugs modulating signal transduction pathways on DEP-induced IL-8 mRNA expression. BEAS-2B cells were treated with each of the drugs 1 h before (a) DEPs (25 µg/ml) or (b) benzene extracts (dose equivalent to 25 µg/ml of DEPs). The RNA were then isolated after 12 h incubation. The effects of DEPs and benzene extracts were abolished by PDTC, NAC, and SB203580, but not by staurosporin. *P < 0.01, ANOVA. Data are presented as means ± SEM from three separate experiments.

Effects of DEP-Derived Components on Cytokine/ Chemokine Release by BEAS-2B and Peripheral Airway Epithelial Cells

Among the five components, benzene components showed a significant stimulatory effect on the release of IL-8, GM-CSF, and RANTES by BEAS-2B and peripheral airway epithelial cells, as shown in Tables 1 and 2. The other components, including n-hexane, chloroform, ethyl acetate and methanol extracts, partially showed a significant stimulatory effect on some of the cytokine/chemokine release.

Benzene-Extracted Components Activated NF-B Binding Activity, as Assessed by EMSA

Because it has been shown that nuclear transcription factor NF-B plays an important role in the transcriptional regulation of IL-8 gene expression (20, 21), and because we have shown that DEP-induced IL-8 mRNA was via the activation of NF-B (18), we attempted to evaluate by EMSA the effect of DEP-derived components on NF-B activation in human bronchial epithelial cells. The cells were treated with DEPs and their components (equivalent to 25 µg/ml of DEPs) for 4 h, and the nuclear extracts were isolated for EMSA as described in MATERIALS AND METHODS. Benzene components as well as DEPs increased the NF-B binding to the labeled oligonucleotide double-stranded DNA (Figure 4). The specificity of the binding was ascertained by the supershift of the bands with antibodies to p65 and p50 as well as the reduced intensity of the signals with excess amounts (×100) of cold DNA NF-B probes (Figure 4).

View larger version (91K): [in this window] [in a new window]   Figure 4.   Effects of DEPs and their components on NF-B activation in human bronchial epithelial cells as assessed by EMSA. The cells were treated with DEPs (25 µg/ml) or each of five components for 4 h, and the nuclear extracts were isolated for EMSA assay as described in MATERIALS AND METHODS. Benzene extracts as well as DEPs increased the nuclear binding to the labeled oligonucleotide double-stranded DNA. The specificity of the binding was ascertained by the supershift of the bands with antibodies to p65 and p50 as well as the reduced intensity of the signals with excess amounts (×100) of cold DNA probes. Among the other components, chloroform and ethyl acetate extracts induced a weakly increased NF-B binding to the specific oligonucleotides. (): vehicle alone.

BaP Increased IL-8 mRNA Expression and Release of Several Cytokines in Human Bronchial Epithelial Cells

Finally, we checked the activity of BaP, an important compound of benzene-extracted substances. BaP significantly increased IL-8 mRNA levels in BEAS-2B, as shown in Figures 5a and 5b. This activity of BaP was suppressed by the preincubation with PDTC, NAC, and SB203580, but not with staurosporin. This compound also increased the release of IL-8, GM-CSF, and RANTES (Figure 5c).

View larger version (23K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 5.   Effects of BaP on human bronchial epithelial cells. (a) A representative result by Northern blot analysis showing the increased IL-8 mRNA levels by BaP. Pretreatment with PDTC, NAC, and SB203580 showed a decreased level of IL-8 mRNA (BaP at 50 µg/ml). (b) Data are presented as means ± SEM from three different experiments. *P < 0.05 compared with baseline, ANOVA. (c) BaP showed a dose-dependent stimulatory effect on IL-8, GM-CSF, and RANTES release from BEAS-2B cells (baseline release = 100% in each release). *P < 0.05 compared with baseline, ANOVA.

	Discussion

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For the present report, we studied the activities of DEP components on the expression of IL-8 mRNA as well as on release of several cytokines/chemokines by human bronchial epithelial cells. The results are summarized as follows: (1) The levels of IL-8 mRNA significantly increased only by benzene-extracted components among others, as assessed by Northern blot analysis. (2) Benzene components alone showed a significant stimulatory effect on the release of all of IL-8, RANTES, and GM-CSF, although some other components partially stimulated the release of cytokines/ chemokines by bronchial epithelial cells. (3) Among several signal blockers, PDTC, NAC, and SB203580, but not staurosporin, suppressed the actions of DEPs. These findings were generally in agreement with previous reports in different cell systems or stimuli (22, 23). Importantly, the activity of benzene extracts was also abolished in the similar fashion. (4) Benzene extracts also induced NF-B activation, which is thought to be crucial for IL-8 gene expression (18, 20, 21). (5) We further studied the activity of BaP, one of the putative chemicals in benzene-extracted components, on human bronchial epithelial cells. This compound was shown to stimulate epithelial cells to upregulate IL-8 mRNA expression and release of cytokines/ chemokines in vitro.

DEPs have now been postulated to induce intense inflammatory reactions in the airways. Sagai and colleagues (4) showed that intratracheal instillation of DEPs induced airway inflammatory changes associated with hyperresponsiveness and cell infiltration, such as eosinophils in mice, which mimic those found in bronchial asthma. Salvi and coworkers (8) demonstrated that short-term (1 h) exposure to diesel exhaust in healthy volunteers induced acute inflammatory responses in the lower respiratory tract, as assessed by airway lavage and mucosal biopsy. It is quite likely that these biologic responses to DEPs were via the actions of cytokines, chemokines, and other inflammatory mediators locally produced in the airways. Airway epithelial cells are the first cells that contact a variety of exogenous agents, including DEP, and it is now clear that these cells have a potential to express and release a variety of cytokines and chemokines important in the airway inflammatory responses (24).

In the previous reports (7, 18), we found that DEPs stimulated IL-8 gene expression and IL-8 production in human bronchial epithelial cells. This effect was largely due to increased transcriptional rates with no apparent change in the decay process of the mRNA (18). Studies with EMSAs suggested that DEPs induced the activation of the transcription factor NF-B, which is considered to play an important role in the gene regulation of IL-8. Treatment of BEAS-2B cells with DEPs that were transfected with the normal B-like sites resulted in a significant increase in luciferase activity, whereas the cells with mutated B-like sites showed no response to IL-1 and DEPs, indicating that DEP-induced increased NF-B binding to its motif was actually important in increased IL-8 gene expression in BEAS-2B cells (18).

It is generally considered difficult to determine exactly the dosages of DEPs and their components that actually reach the airways and affect the bronchial epithelium. Previous reports showed that people are exposed to a dose of 0.2 mg of DEPs in a period of 1 d in Los Angeles (25). Many in vitro studies (9) have used DEPs at the ranges of 0 to 100 µg/ml. As Bayram and colleagues pointed out, in vitro studies have several limitations, because natural defense mechanisms in vivo are absent and the particles, once suspended, may alter (9). We have recently developed a system that enables the cells to be exposed to freshly generated diesel exhaust (26). Studies using this system may better replicate activities of fresh DEPs, but it was impossible to evaluate the activity of each extract by this system.

It seems important to find the responsible components or chemicals for more effective environmental control in the atmosphere for lung health. In the present studies, we attempted to determine what components were important in the activities of DEPs in vitro. Boland and associates (11) recently found that these cells are capable of phagocytosing the particles, as evaluated by fluorescent-bound particles and flow cytometry. They further indicated that use of oxidation catalyst, which reduced the polyaromatic hydrocarbons (PAHs) and nitro-PAH contents of DEPs by 50 to 60%, markedly diminished the release of cytokines induced by DEPs. DEPs contain a vast number of organic molecules on their surfaces, and the findings of Boland and associates strongly suggested that adsorbed organic compounds such as PAHs are important for the biological activities of DEPs. Kumagai and coworkers (13) studied the activity of components in terms of production of reactive oxygen species, and found that quinones in methanol extracts were mainly responsible for that activity. In the present study, we could clearly show that the components extracted by benzene from DEPs were largely responsible for the activities of cytokine production on airway epithelial cells.

In the present study, we chose BaP to study its potential to express and release cytokines because it has generally been considered to be one of the important hydrocarbons for lung health (27). DEPs contain compounds having three to five benzene rings which are classified as PAHs. The most common PAHs contained in DEPs are phenanthrenes (52%), fluorenes (15%), naphthalens (13%), fluoranthrenes (19%), and pyrenes (10%), including benzopyrene (29, 30). Among those, the pyrenes extracted from DEPs had the same stimulatory effect on IgE production in mice (31). We have analyzed the relative amounts of BaP contained in each extract by high-performance liquid chromatography (unpublished data). Approximately 82% of the total BaP in DEPs was contained in benzene extracts, 5% in n-hexane extracts, 8% in chloroform, and 5% in ethyl acetate. We agree that the concentrations of BaP used in the present study were higher than the actual concentrations contained in DEPs, but these dosages were comparable with those of beta-naphthoflavone, another representative PAH, used in the report by Ng and associates (32). BaP and other pyrenes are hardly soluble in media even with DMSO, and the actual dosages in the experiments might be less than the calculated dosage.

It should also be emphasized that other substances in benzene extracts might be important. We do not argue that BaP was the sole causative substance in benzene-extracts, but speculate that some other compounds were also involved. We also suggest that other components contained in other extracts might also be responsible for the bioactivities of DEPs. Components other than benzene extracts showed some tendency to inhibit IL-8 mRNA by Northern blot analysis or NF-B activation, but not significantly. In contrast, some of the other components showed weak but significant stimulatory effect on cytokine release (Tables 1 and 2). The reasons for such apparent discrepancies between the gene expression and protein release were unclear. It was probable that multiple agents in DEPs might be responsible, presumably positively and negatively, for its activity at several steps from transcriptional to secretory regulation. The transcriptional regulation might be better understood by reporter gene assay.

The specific receptor for PAHs such as BaP, AhR, is widely expressed in human tissues (33). Recently, Nel and colleagues (29) and Ng and associates (32) showed that a representative PAH, beta-naphthoflavone, activated Jun-N-terminal kinase and p38MAPK activities in parallel with the generation of activator protein-1 mobility shift complexes in THP-1 and RAW264.7 macrophage cell lines. They also showed that the activation of the MAPKs was dependent on generation of oxidative stress, because that could be inhibited by NAC. Although the molecular events linking the activation of the MAPK pathways and NF-B activation remain unclear, DEP-induced production of intracellular oxidants might be involved in these processes (34). In fact, Lim and coworkers (35) showed that reactive oxygen species such as superoxide and nitric oxide were produced by DEP treatment in airway epithelial cells. The activity to stimulate cytokine production of benzene extracts as well as DEPs was inhibited by NAC, PDTC, and SB203580, suggesting that oxidants and p38MAPK pathways are involved in this process. It seems worthy of pursuing biologic activities and following intracellular molecular events elicited by BaP and related molecules adsorbed onto DEPs to better understand the influences of these particulate matters in human lung health.