Organic Extracts Of Diesel Exhaust Particles Research Articles

The pro-inflammatory effects of combined exposure to diesel exhaust particles and mineral particles in human bronchial epithelial cells

BackgroundPeople are exposed to ambient particulate matter (PM) from multiple sources simultaneously in both environmental and occupational settings. However, combinatory effects of particles from different sources have received little attention in experimental studies. In the present study, the pro-inflammatory effects of combined exposure to diesel exhaust particles (DEP) and mineral particles, two common PM constituents, were explored in human lung epithelial cells.MethodsParticle-induced secretion of pro-inflammatory cytokines (CXCL8 and IL-1β) and changes in expression of genes related to inflammation (CXCL8, IL-1α, IL-1β and COX-2), redox responses (HO-1) and xenobiotic metabolism (CYP1A1 and CYP1B1) were assessed in human bronchial epithelial cells (HBEC3-KT) after combined exposure to different samples of DEP and mineral particles. Combined exposure was also conducted using lipophilic organic extracts of DEP to assess the contribution of soluble organic chemicals. Moreover, the role of the aryl hydrocarbon receptor (AhR) pathway was assessed using an AhR-specific inhibitor (CH223191).ResultsCombined exposure to DEP and mineral particles induced increases in pro-inflammatory cytokines and expression of genes related to inflammation and redox responses in HBEC3-KT cells that were greater than either particle sample alone. Moreover, robust increases in the expression of CYP1A1 and CYP1B1 were observed. The effects were most pronounced after combined exposure to α-quartz and DEP from an older fossil diesel, but enhanced responses were also observed using DEP generated from a modern biodiesel blend and several stone particle samples of mixed mineral composition. Moreover, the effect of combined exposure on cytokine secretion could also be induced by lipophilic organic extracts of DEP. Pre-incubation with an AhR-specific inhibitor reduced the particle-induced cytokine responses, suggesting that the effects were at least partially dependent on AhR.ConclusionsExposure to DEP and mineral particles in combination induces enhanced pro-inflammatory responses in human bronchial epithelial cells compared with exposure to the individual particle samples. The effects are partly mediated through an AhR-dependent pathway and lipophilic organic chemicals in DEP appear to play a central role. These possible combinatory effects between different sources and components of PM warrant further attention and should also be considered when assessing measures to reduce PM-induced health effects.

Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals

Diesel exhaust particles (DEPs) are a major component of diesel emissions, responsible for a large portion of their toxicity. In this study, we examined the toxic effects of DEPs on endothelial cells and the role of DEP-induced heme oxygenase-1 (HO-1) expression. Human microvascular endothelial cells (HMECs) were treated with an organic extract of DEPs from an automobile engine (A-DEP) or a forklift engine (F-DEP) for 1 and 4h. ROS generation, cell viability, lactate dehydrogenase leakage, expression of HO-1, inflammatory genes, cell adhesion molecules and unfolded protein respone (UPR) gene were assessed. HO-1 expression and/or activity were inhibited by siRNA or tin protoporphyrin (Sn PPIX) and enhanced by an expression plasmid or cobalt protoporphyrin (CoPPIX). Exposure to 25μg/ml of A-DEP and F-DEP significantly induced ROS production, cellular toxicity and greater levels of inflammatory and cellular adhesion molecules but to a different degree. Inhibition of HO-1 enzymatic activity with SnPPIX and silencing of the HO-1 gene by siRNA enhanced DEP-induced ROS production, further decreased cell viability and increased expression of inflammatory and cell adhesion molecules. On the other hand, overexpression of the HO-1 gene by a pcDNA 3.1D/V5-HO-1 plasmid significantly mitigated ROS production, increased cell survival and decreased the expression of inflammatory genes. HO-1 expression protected HMECs from DEP-induced prooxidative and proinflammatory effects. Modulation of HO-1 expression could potentially serve as a therapeutic target in an attempt to inhibit the cardiovascular effects of ambient PM.

Reactive Oxygen Species- and Nitric Oxide-Mediated Lung Inflammation and Mitochondrial Dysfunction in Wild-Type and iNOS-Deficient Mice Exposed to Diesel Exhaust Particles

Pulmonary responses to diesel exhaust particles (DEP) exposure are mediated through enhanced production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM). The current study examined the differential roles of ROS and NO in DEP-induced lung injury using C57B/6J wild-type (WT) and inducible NO synthase knockout (iNOS KO) mice. Mice exposed by pharyngeal aspiration to DEP or carbon black particles (CB) (35 mg/kg) showed an inflammatory profile that included neutrophil infiltration, increased lactate dehydrogenase (LDH) activity, and elevated albumin content in bronchoalveolar lavage fluid (BALF) at 1, 3, and 7 d postexposure. The organic extract of DEP (DEPE) did not induce an inflammatory response. Comparing WT to iNOS KO mice, the results show that NO enhanced DEP-induced neutrophils infiltration and plasma albumin content in BALF and upregulated the production of the pro-inflammatory cytokine interleukin 12 (IL-12) by AM. DEP-exposed AM from iNOS KO mice displayed diminished production of IL-12 and, in response to ex vivo lipopolysaccharide (LPS) challenge, decreased production of IL-12 but increased production of IL-10 when compared to cells from WT mice. DEP, CB, but not DEPE, induced DNA damage and mitochondria dysfunction in AM, however, that is independent of cellular production of NO. These results demonstrate that DEP-induced immune/inflammatory responses in mice are regulated by both ROS- and NO-mediated pathways. NO did not affect ROS-mediated mitochondrial dysfunction and DNA damage but upregulated IL-12 and provided a counterbalance to the ROS-mediated adaptive stress response that downregulates IL-12 and upregulates IL-10.

Suppression of Phagocytic and Bactericidal Functions of Rat Alveolar Macrophages by the Organic Component of Diesel Exhaust Particles

Exposure to diesel exhaust particles (DEP) was shown to increase the susceptibility of the lung to bacterial infection in rats. In this study, the effects of DEP on alveolar macrophage (AM) phagocytic and bactericidal functions and cytokine secretion by AM and lymphocytes in response to Listeria monocytogenes infection were investigated in vitro and the roles of different DEP components in these processes were compared. Exposure to DEP or the organic extracts of DEP (eDEP) significantly decreased the phagocytosis and killing of L. monocytogenes by AM obtained from normal rats. Washed DEP (wDEP) also decreased AM phagocytosis and bacterial killing to a lesser extent, whereas carbon black (CB) reduced AM phagocytosis but had no significant effect on AM bactericidal activity. DEP or eDEP concentration-dependently suppressed L. monocytogenes-induced secretion of tumor necrosis factor-α, interleukin (IL)-1β, and IL-12 by AM and of IL-2 and interferon-γ by lymphocytes obtained from L. monocytogenes-infected rats, but augmented the AM secretion of IL-10. wDEP or CB, however, exerted little or no effect on these L. monocytogenes-induced cytokines. These results provide direct evidence that DEP, through the actions of organic components, suppresses AM phagocytic and bactericidal functions in vitro. Inhibition of AM phagocytic function and alterations of AM and lymphocyte cytokine secretion by DEP and DEP organic compounds may be implicated in the diminished AM bactericidal activity and the lymphatic arm of the host immune system, thus resulting in an suppressed pulmonary clearance of L. monocytogenes and an increased susceptibility of the lung to bacterial infection.

Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles

Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 microg/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 microg/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-alpha or 0.5 ng/ml transforming growth factor-beta1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells.

Effects of exposure to diesel exhaust particles (DEP) on pulmonary metabolic activation of mutagenic agents

Exposure of rats to diesel exhaust particles (DEP) or carbon black (CB) has been shown to induce time-dependent changes in CYP1A1and CYP2B1 in the lung. The present study evaluated the role of these metabolic enzymes on the pulmonary bioactivation of mutagens. Male Sprague–Dawley rats were intratracheally instilled with saline (control), DEP or CB (35 mg/kg body weight) and sacrificed at 1, 3, or 7 days post-exposure. Both control and exposed lung S9 increased the mutagenic activity of 2-aminoanthracene (2-AA), 2-aminofluorene (2-AF), 1-nitropyrene (1-NP), and the organic extract of DEP (DEPE) in Ames tests with Salmonella typhimurium YG1024 in a dose-dependent manner. Lung microsomes prepared form control or particle-exposed S9, but not cytosolic protein, activated 2-AA mutagenicity. Compared to saline controls, CB-exposed S9 was a less potent inducer of 2-AA mutagenicity at all time points, whereas DEP-exposed S9 was less potent than control saline at 3 and 7 days but not 1 day post-exposure. At 3 days post-exposure, DEP- or CB-exposed lung S9 did not significantly affect the mutagenicity of DEPE or 1-NP, when compared to the controls. The mutgenicity of 2-AA, 2-AF, 1-NP, and DEPE were significantly decreased in the presence of inhibitors for CYP1A1 (α-naphthoflavone) or CYP2B (metyrapone), but markedly enhanced by CYP1A1 or CYP2B1 supersomes with all the cofactors, suggesting that both CYP1A1 and CYP2B1 were responsible for mutagen activation. These results demonstrated that exposure of rats to DEP or CB altered metabolic activity of lung S9 and S9 metabolic activity dependent mutagen activation. The bioactivation of mutagens are metabolic enzyme- and substrate-specific, and both CYP1A1 and CYP2B1 play important roles in pulmonary mutagen activation.

CDNA microarray analysis of rat alveolar epithelial cells following exposure to organic extract of diesel exhaust particles

Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis. Comprehensive evaluation is required to know the mechanisms underlying the effects of air pollutants including DEP on lung diseases. Using a cDNA microarray, we examined changes in gene expression in SV40T2 cells, a rat alveolar type II epithelial cell line, following exposure to an organic extract of DEP. We identified candidate sensitive genes that were up- or down-regulated in response to DEP. The cDNA microarray analysis revealed that a 6-h exposure to the DEP extract (30 μg/ml) increased (>2-fold) the expression of 51 genes associated with drug metabolism, antioxidation, cell cycle/proliferation/apoptosis, coagulation/fibrinolysis, and expressed sequence tags (ESTs), and decreased (<0.5-fold) that of 20 genes. In the present study, heme oxygenase (HO)-1, an antioxidative enzyme, showed the maximum increase in gene expression; and type II transglutaminase (TGM-2), a regulator of coagulation, showed the most prominent decrease among the genes. We confirmed the change in the HO-1 protein level by Western blot analysis and that in the enzyme activity of TGM-2. The organic extract of DEP increased the expression of HO-1 protein and decreased the enzyme activity of TGM-2. Furthermore, these effects of DEP on either HO-1 or TGM-2 were reduced by N-acetyl- l-cysteine (NAC), thus suggesting that oxidative stress caused by this organic fraction of DEP may have induced these cellular responses. Therefore, an increase in HO-1 and a decrease in TGM-2 might be good markers of the biological response to organic compounds of airborne particulate substances.

Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells.

Diesel exhaust particles (DEP) induce a proinflammatory response in human bronchial epithelial cells (16HBE) characterized by the release of proinflammatory cytokines after activation of transduction pathways involving MAPK and the transcription factor NF-kappaB. Because cellular effects induced by DEP are prevented by antioxidants, they could be mediated by reactive oxygen species (ROS). Using fluorescent probes, we detected ROS production in bronchial and nasal epithelial cells exposed to native DEP, organic extracts of DEP (OE-DEP), or several polyaromatic hydrocarbons. Carbon black particles mimicking the inorganic part of DEP did not increase ROS production. DEP and OE-DEP also induced the expression of genes for phase I [cytochrome P-450 1A1 (CYP1A1)] and phase II [NADPH quinone oxidoreductase-1 (NQO-1)] xenobiotic metabolization enzymes, suggesting that DEP-adsorbed organic compounds become bioavailable, activate transcription, and are metabolized since the CYP1A1 enzymatic activity is increased. Because NQO-1 gene induction is reduced by antioxidants, it could be related to the ROS generated by DEP, most likely through the activation of the stress-sensitive Nrf2 transcription factor. Indeed, DEP induced the translocation of Nrf2 to the nucleus and increased protein nuclear binding to the antioxidant responsive element. In conclusion, we show that DEP-organic compounds generate an oxidative stress, activate the Nrf2 transcription factor, and increase the expression of genes for phase I and II metabolization enzymes.

Oxidative-stress potency of organic extracts of diesel exhaust and urban fine particles in rat heart microvessel endothelial cells

Exposure to fine particulate materials is associated with an increase in mortality rate of cardiovascular diseases. Particles deposited in the lung may affect the vascular system both directly (leaching of soluble components from particles) and indirectly (via cytokines and mediators). The present study addressed cytotoxicity and oxidative stress potency of organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) in rat heart microvessel endothelial (RHMVE) cells. The LC 50 values of OE-DEP and OE-UFP were calculated to be 17 and 34 μg/ml, respectively, suggesting that OE-DEP was more cytotoxic than OE-UFP. The viability of OE-DEP- and OE-UFP-exposed cells was ameliorated by N-acetyl- l-cysteine (NAC). The cell monolayer was exposed to 0 (control), 1, 3, and 10 μg/ml OE-DEP for 6 h and mRNA levels of antioxidant enzymes such as heme oxygenase-1 (HO-1), thioredoxin peroxidase 2 (TRPO), glutathione S-transferase P subunit (GST-P), and NADPH dehydrogenase (NADPHD) were quantitated by northern analysis. All those mRNA levels increased dose-dependently with OE-DEP and HO-1 mRNA showed the most marked response to OE-DEP. mRNA levels of those antioxidant enzymes and heat shock protein 72 (HSP72) in OE-DEP-exposed cells were higher than those of OE-UFP-exposed cells as compared at the same concentration. The transcription levels of HO-1 and HSP72 in OE-DEP- and OE-UFP-exposed cells were also reduced by NAC. Those results suggest that the organic fraction of particulate materials in the urban air has a potency to cause oxidative stress to endothelial cells and may be implicated in cardiovascular diseases through functional changes of endothelial cells.

Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils.

An epidemiologic relationship between airway allergic diseases and exposure to atmospheric pollutants has been demonstrated and suggested to be one factor in the increasing prevalence of asthma. Diesel exhaust particles (DEPs) have been shown to participate in the development of allergic airway inflammation, in which the targets include macrophages, B and T cells, epithelial cells, and mast cells. In addition to the adjuvant effect of DEPs on total and allergen-specific IgE production, DEPs also act to induce chemokines and cytokines and may play a key role in primary sensitization. DEPs have been shown to increase local IL-4-containing Kit(+) cells soon after in vivo nasal challenge. The aim of this study was to examine the effects of DEPs on human basophils, a key source of IL-4. Peripheral blood leukocytes from allergic and control subjects were cultured in the presence of organic extracts of DEP (DEPex) with or without allergen. The cultures were analyzed for IL-4-containing cells by using multiparameter flow cytometry, IL-4 secretion with ELISA, and histamine release. Basophils, when exposed in vitro to DEPex, expressed IL-4 and released histamine significantly (P <.01) more than with antigen activation. DEPex did not synergize with allergen in cytokine production and histamine release. DEPex-induced basophil IL-4 expression peaked at 2 hours and persisted through 20 hours, in contrast to allergen-induced IL-4, which was transient. The effect of DEPex on basophil cytokine expression and histamine release was dose dependent and occurred with cells from both allergic and nonallergic subjects. DEPex induced IL-4 expression and histamine release in highly enriched basophil populations, suggesting it acts directly on basophils. Other peripheral blood leukocytes, including T cells, did not contribute to this cytokine expression. Preincubation with N-acetylcysteine completely abrogated DEPex-driven basophil IL-4 expression. Basophils are a direct target for DEPex, inducing IL-4 expression and histamine release in an IgE-allergen independent fashion. N-acetylcysteine inhibition of DEPex-driven IL-4 expression provides evidence that generation of reactive oxygen species is required for the effects observed. The capability of DEPex to activate basophils in both allergic and nonallergic subjects suggests a potential role of this pollutant in the increasing prevalence of allergic diseases.

Diesel exhaust particles induce an inflammatory response in airway epithelial cells: involvement of reactive oxygen species.

Epidemiological studies have shown that high particulate air pollution is associated with an increase in hospital admissions for respiratory diseases and with an exacerbation of asthma symptoms [8]. In urban areas, traffic appears to be the main source of particulate airborne pollutants. Diesel engine powered cars, which produce the most abundant components of PM2.5 (Particulate Matter 2.5 µm), are suspected to be responsible for this exacerbation of respiratory diseases but their real impact on health has not been fully elucidated. Recent experimental studies have emphasized the role of diesel exhaust particles (DEP) in the development of an inflammatory response. Nasal challenges of humans with DEP result in the local increase in IgE and cytokine production [7]. In vitro studies have shown that airway epithelial cells, one of the main cellular targets of DEP, directly participate in this inflammatory response [2]. Indeed, cultured human nasal and bronchial epithelial cells exposed to DEP released in a time and dose dependent process, proinflammatory cytokines such as GM-CSF and IL-8 known to be involved in allergic diseases [3]. This increased release of GM-CSF has been shown to be subsequent to transcription [4]. Since airway epithelial cells can be effectors of the inflammatory response, it is necessary to investigate the cellular and molecular mechanisms leading to this response and to determine the respective role of DEP components in these mechanisms. DEP are composed of a carbonaceous core with adsorbed organic compounds (polyaromatic hydrocarbons (PAH), nitroaromatic hydrocarbons, quinones, aldehydes and aliphatic hydrocarbons) which could represent up to 60% of the mass of the particle depending on the vehicle and the fuel composition. The cellular and molecular events induced by DEP were investigated using a model of bronchial epithelial cells, the human bronchial epithelial cell line (16HBE 14 o-) and standard reference DEP (SRM 1650). The effects of native DEP were compared to those induced by the organic extracts of DEP (OE-DEP) obtained by extraction of DEP with dichloromethan and by the carbonaceous core represented either by carbon black particles or stripped DEP (sDEP) recovered after extraction. DEP trigger MAPK Erk 1/2 signalling pathways as a specific inhibitor of this pathway (PD 98059) clearly reduced the DEP-induced GM-CSF release. Moreover the phosphorylated form of Erk 1/2

Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression.

Diesel exhaust particles (DEP) are known to enhance inflammatory responses in human volunteers. In cultured human bronchial epithelial (16HBE) cells, they induce the release of proinflammatory cytokines after triggering transduction pathways, including nuclear factor (NF)-kappaB activation and mitogen-activated protein kinase (MAPK) phosphorylation. This study compares the effects of native DEP (nDEP), organic extracts of DEP (OE-DEP), and carbonaceous particles, represented by stripped DEP (sDEP) and carbon black particles (CB), in order to clarify their respective roles. OE-DEP and nDEP induce granulocyte macrophage colony-stimulating factor (GM-CSF) release, NF-kappaB activation, and MAPK phosphorylation. The carbonaceous core generally induces less intense effects. Reactive oxygen species are produced in 16HBE cells and are involved in GM-CSF release and in the stimulation of NF-kappaB DNA binding by nDEP and OE-DEP. We demonstrate, for the first time, in airway epithelial cells in vitro that nDEP induce the expression of the CYP1A1, a cytochrome P450 specifically involved in polycyclic aromatic hydrocarbons metabolism, thereby demonstrating the critical role of organic compounds in the DEP-induced proinflammatory response. Understanding the respective contributions of DEP components in these effects is important for vehicle manufacturers in order to improve their exhaust gas post-treatment technologies. In conclusion, the DEP-induced inflammatory response in airway epithelial cells mainly involves organic compounds such as PAH, which induce CYP1A1 gene expression.

Activation of Transcription Factors by Diesel Exhaust Particles in Human Bronchial Epithelial Cells in Vitro

Diesel exhaust particles (DEP) are suspected to be involved in the aggravation of inflammatory respiratory diseases. We have shown previously, in human bronchial epithelial cell line 16HBE 14o-, that DEP induced the release of the proinflammatory cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8) after 24 h of exposure. Gene expression of these cytokines is regulated by transcription factors including NF-κB and AP-1, which are known to be sensitive to oxidative stress. Their activation by DEP was investigated in comparison with a pure oxidant, H2O2 A 4-h exposure to DEP (10 μg/cm2) or to H2O2 (100 µM) increased NF-κB DNA binding in 16HBE cells as assessed by electrophoretic mobility shift assay. AP-1 was only activated by H202 in the same conditions. Organic extracts of DEP increased NF-κB DNA binding as did native DEP, suggesting the role of the polycyclic aromatic hydrocarbons (PAH) in this NF-κB increased DNA binding. Dimethylthiourea (DMTU), an antioxidant, inhibited the NF-κB DNA binding induced by DEP, suggesting an involvement of reactive oxygen species (ROS) in the transduction pathways leading to NF-κB activation. Moreover, the MEK pathway inhibitor PD98059 inhibited DEP-induced NF-κB DNA binding. The role of Erk 1/2 was likely implicated, since DEP induced an increase of Erk phosphorylation.

The Organic Component of Diesel Exhaust Particles and Phenanthrene, a Major Polyaromatic Hydrocarbon Constituent, Enhances IgE Production by IgE-Secreting EBV-Transformed Human B Cellsin Vitro

Suspended airborne particulate matter such as diesel exhaust particles (DEP) have been implicated in the increased incidence of respiratory allergic diseases that has occurred over the past century. Studiesin vitroandin vivohave shown that DEP may enhance allergic antibody (IgE) expression. DEP contain a wide spectrum of polycyclic aromatic hydrocarbons (PAH) that have been reported to have direct effects on the immune system, including the modulation of IgE production using various human and murine cell populations. We investigated the effects of the organic extract of DEP (PAH-DEP) and particularly, phenanthrene, a major component of DEP,in vitroon IgE production by 2C4/F3, a human Epstein–Barr virus transformed isotype switched, IgE producing B cell line. Phenanthrene consistently enhanced 2C4/F3 IgE production two- to threefold. Thisin vitroenhancement was associated with an increased expression of total IgE mRNA. Furthermore, the pattern of mRNA's coding for distinct isoforms of the epsilon chain was altered by both DEP-PAH and phenanthrene. While phenanthrene increased the level of productive epsilon transcripts, it did not increase epsilon germ line transcription. These effects were not due to an alteration of the cell cycle. Unstimulated 2C4/F3 cells contained detectable mRNA for IL6, IL10, TNF-α, and interestingly IL4; however, addition of PAH-DEP or phenanthrene did not significantly alter the level of these cytokines and thus did not appear to account for our findings. Thus, we have used ourin vitromodel to dissect the mechanism of DEP-PAH on IgE production in postswitch IgE producing cells and shown that phenanthrene, an important component in DEP and other pollutants, can act in a similar manner.

Effects of Diesel Exhaust Particles on the Release of Interleukin-1 and Tumor Necrosis Factor-Alpha from Rat Alveolar Macrophages

The effects of diesel exhaust particles (DEP) and their components (washed dust and methanol extracts) on the release of proinflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-α) by alveolar macrophages (AM) were investigated. Rat AM were incubated with 0, 5, 10, 20, 50, or 100 μg/10−6 AM/mL of DEP, methanol-washed DEP, or equivalent concentrations of DEP methanol extracts at 37°C for 24 h. AM-conditioned supernatants were collected and assayed for the activities of IL-1 and TNF-α. At high concentrations, both DEP and DEP methanol extracts were shown to increase IL-1-like activity secreted by AM, whereas methanol-washed DEP had no effect. Neither DEP, methanol-washed DEP, nor DEP methanol extracts was found to stimulate the secretion of TNF-α. The effects of DEP on the release of IL-1 and TNF-α by lipopolysaccharide (LPS)- or interferon-gamma (IFN-γ)-primed AM were also studied. AM were preincubated with various concentrations of DEP for 2 h, then challenged with either 0.1 μg/mL of LPS or 5 units/mL of IFN-γ. DEP inhibited LPS-stimulated production of IL-1 and TNF-α. These inhibitory effects were attributed to the organic extracts of DEP. In contrast, stimulation of AM production of TNF-α by IFN-γ was not affected by DEP exposure. In summary, evidence that DEP enhanced the production of IL-1 by AM in vitro suggests that this proinflammatory cytokine may play a role in the pulmonary response to DEP inhalation. The suppressive response of DEP-pretreated AM to LPS stimulation may be a contributing factor to the impairment of pulmonary defense system after prolonged DEP exposure.

Isolation, identification and bacterial mutagenicity of 2-nitro-9-fluorenone from diesel-exhaust particle extracts

Organic extracts of diesel-exhaust particles show direct mutagenic activity in the Salmonella typhimurium bacterial mutagenicity assay. Nitro-aromatic compounds are believed to be responsible for part of the mutagenicity. A previously unidentified polyfunctional nitro-aromatic compound, 2-nitro-9-fluorenone (2N-Fone) was isolated from diesel-exhaust particles using a two-step fractionation scheme consisting of Sephadex LH20 chromatography and silica-gel thin-layer chromatography. Positive identification was by gas chromatography/mass spectroscopy and coelution with an authentic standard. Direct and indirect mutagenicities of 2N-Fone in several bacterial strains were also determined. The results indicated that 2N-Fone produces 60–70 rev/nmole of direct mutagenic activity, and is about 1 5 to 1 10 as mutagenic as 1-nitropyrene.