Compounds Of Diesel Exhaust Particles Research Articles

Effects of organic and inorganic compounds of diesel exhaust particles on the mucociliary epithelium: An experimental study on the frog palate preparation

The toxic actions of acute exposition to different diesel exhaust particles (DEPA) fractions on the mucociliary epithelium are not yet fully understood due to different concentrations of organic and inorganic elements. These chemicals elements produce damage to the respiratory epithelium and exacerbate pre-existent diseases. In our study we showed these differences in two experimental studies. Study I (dose-response curve - DRCS): Forty frog-palates were exposed to the following dilutions: frog ringer, intact DEPA diluted in frog-ringer at 3mg/L, 6mg/L and 12mg/L. Study II (DEPF) (DEPA fractions diluted at 12mg/L): Fifty palates - Frog ringer, intact DEPA, DEPA treated with hexane, nitric acid and methanol. Variables analyzed: relative time of mucociliary transport (MCT), ciliary beating frequency (CBF) and morphometric analysis for mucin profile (neutral/acid) and vacuolization. The Results of DRCS: Group DEPA-12mg/L presented a significant increase in the MCT (p<0.05), proportional volume of acid mucus (p<0.05) and decreased proportional volume of neutral mucus and vacuoles (p<0.05). In relation of DEPF: A significant increase in the MCT associated to a decrease in the proportional volume of neutral mucus was founded in nitric acid group. In addition, a significant increase in the proportional volume of acid mucus was found in methanol group. We concluded that: 1) Increasing concentrations of intact DEPA can progressively increase MCT and promote an acidification of intra-epithelial mucins associated to a depletion of neutral mucus. 2) Intact DEPA seem to act as secretagogue substance, promoting mucus extrusion and consequently reducing epithelial thickness. 3) Organic fraction of low polarity seems to play a pivotal role on the acute toxicity to the mucociliary epithelium, by promoting a significant increase in the MCT associated to changes in the chemical profile of the intracellular mucins.

Ex vivo effects of naphthoquinones on allergen-sensitized mononuclear cells in mice

Naphthoquinone (NQ), one of the extractable chemical compounds of diesel exhaust particles, enhances allergic asthma traits in mice. However, it remains unknown whether: (1) several types of NQs have the same potential to facilitate allergies; and (2) NQs synergistically disrupt the functional phenotypes of immune cells. The aim of the present study was to investigate the effects of two types (1,2- and 1,4-) of NQs on sensitized mononuclear cells using an ex vivo assay. Male BALB/c mice were repeatedly and intraperitoneally administered ovalbumin (OVA: 20 µg) plus alum with or without two different doses of each NQ. After the final administration, splenocytes (mononuclear cells) were isolated from these mice and cultured in the presence of OVA. Helper T-related cytokines in the culture supernatants and downstream molecules were then evaluated. Protein levels of interferon-γ were higher in the supernatants from 1,2-NQ and 1,4-NQ at low dose + OVA-exposed mononuclear cells following the OVA stimulation than in those from OVA-exposed mononuclear cells. Interleukin (IL)-13 levels were higher in the supernatants from low dose NQs + OVA-exposed mononuclear cells. IL-17 levels were significantly higher in the supernatants from low dose 1,2-NQ + OVA-exposed mononuclear cells. The quantity of phosphorylated STAT6 in the nuclei of these cells was significantly greater in the low dose NQ + OVA groups than in the OVA group. These findings suggest NQs differently enhance allergen sensitization in the context of the Th response against mononuclear cells such as lymphocytes.

Enriched inorganic compounds in diesel exhaust particles induce mitogen-activated protein kinase activation, cytoskeleton instability, and cytotoxicity in human bronchial epithelial cells

This study assessed the effects of the diesel exhaust particles on ERK and JNK MAPKs activation, cell rheology (viscoelasticity), and cytotoxicity in bronchial epithelial airway cells (BEAS-2B). Crude DEP and DEP after extraction with hexane (DEP/HEX) were utilized. The partial reduction of some DEP/HEX organics increased the biodisponibility of many metallic elements. JNK and ERK were activated simultaneously by crude DEP with no alterations in viscoelasticity of the cells. Mitochondrial activity, however, revealed a decrease through the MTT assay. DEP/HEX treatment increased viscoelasticity and cytotoxicity (membrane damage), and also activated JNK. Our data suggest that the greater bioavailability of metals could be involved in JNK activation and, consequently, in the reduction of fiber coherence and increase in the viscoelasticity and cytotoxicity of BEAS cells. The adverse findings detected after exposure to crude DEP and to DEP/HEX reflect the toxic potential of diesel compounds. Considering the fact that the cells of the respiratory epithelium are the first line of defense between the body and the environment, our data contribute to a better understanding of the pathways leading to respiratory cell injury and provide evidence for the onset of or worsening of respiratory diseases caused by inorganic compounds present in DEP.

Effects of naphthoquinone on airway responsiveness in the presence or absence of antigen in mice

We have recently demonstrated that naphthoquinone (NQ), one of extractable chemical compounds of diesel exhaust particles (DEP), enhances antigen-related airway inflammation with goblet cell hyperplasia in mice (Inoue et al. in Eur Respir J 209(2):259-267, 2007). Further, NQ has enhanced lung expressions of interleukin (IL)-4 and IL-5. However, the effects of NQ on other cardinal features of asthma have not been completely investigated. The aim of the present study was to evaluate the effects of NQ on airway responsiveness on the model. Vehicle, NQ, ovalbumin (OVA), or NQ + OVA was administered intratarcheally to ICR mice for 6 weeks. Twenty-four hours after the last instillation, lung histology, lung functions such as total respiratory system resistance (R) and Newtonian resistance (R (n)), and protein level of IL-13 and mRNA level for MUC5AC in the lung were examined. Repetitive exposure to NQ aggravated antigen-related lung inflammation. NQ alone enhanced R and R (n) as compared to vehicle without statistical significance. OVA alone or NQ plus OVA showed increases in R and R (n), which was prominent in NQ plus OVA (P < 0.05 vs. vehicle). Combined exposure to NQ and OVA elevated the levels of IL-13 and MUC5AC in the lung as compared with exposure to NQ or OVA alone. These results indicate that NQ can enhance airway hyperresponsiveness in the presence or absence of an antigen. Also, amplified lung expressions of IL-13 and MUC5AC might partly contribute to the deterioration of asthma features by NQ.

Thymic involution produced by diesel exhaust particles and their constituents in mice

Epidemiological studies suggest that diesel exhaust particles (DEP) contribute to an increase in allergic diseases. To assess the effects of DEP on the central immune system, mice were exposed to DEP by intraperitoneal (IP) administration. Exposure to DEP resulted in severe thymic involution accompanied by a reduction in the number of thymocytes, especially in cortical CD4+CD8+ double positive and double negative subsets. Core carbon particles associated with a mixture of chemical compounds in DEP did not appear to be responsible for the DEP-induced thymic involution because carbon graphite does not affect neither the number nor the CD4/CD8 profile of thymocytes. Extraction of DEP by ether, acidic and basic solvents showed that several independent fractions including the neutral ether fraction, which contains polycyclic aromatic hydrocarbons (PAH), induced thymic involution. Among major PAH components of DEP, benzo[b]fluoranthene (BbF), benzo[a]pyrene (BaP), and benzo[k]fluoranthene (BkF) were very potent inducers of thymic involution at an ED50 of less than 100 ng per mouse body. Nonetheless, DEP treatment of mice with targeted disruption of genes encoding the aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), or microsomal epoxide hydolase (mEH) indicated that DEP produced thymic involution even in the absence of PAH-induced AHR/ARNT signal transduction or mEH-mediated PAH catabolism. On the other hand, BaP-mediated thymic involution was completely dependent on AHR, partially dependent on ARNT in T cells, and independent of mEH. These results indicate that DEP-induced thymic involution is mediated both by PAH-AHR/ARNT-dependent and -independent mechanisms.

Organic extract of diesel exhaust particles stimulates expression of Ia and costimulatory molecules associated with antigen presentation in rat peripheral blood monocytes but not in alveolar macrophages

We hypothesized that diesel exhaust particles (DEP) induce the activation of antigen-presenting cells (APC) in lung. The present study was designed to clarify the following about DEP: (1) whether it affects the expression of Ia and B7 molecules in alveolar macrophages (AM) as a mature cell or in peripheral blood monocytes (PBM) as an immature cell, (2) if it affects the antigen-presenting (AP) activity of PBM, (3) what component of DEP is responsible for the effects, and (4) whether the effect of DEP is related to oxidative stress. DEP was extracted with methylene chloride. Cells were exposed to whole DEP, organic extract, or residual particles for 24 h. Cell-surface molecules were measured by flow cytometry. AP activity was assessed by antigen-specific T cell proliferation. Whole DEP or organic extract significantly increased the expression of Ia and B7 molecules on PBM but not on AM. No significant effect of residual particles was observed. A low concentration of organic extract also increased the AP activity of PBM. When the induction of an antioxidative enzyme was assessed, heme oxygenase-1 protein was found to be significantly increased by exposure to whole DEP, and the organic extract was more effective than the residual particles. Furthermore, the organic extract-induced expression of Ia antigen on PBM was reduced by the addition of an antioxidative agent. These results suggest that DEP may act on immature APC and enhance their AP activity and that the action contributing to oxidative stress may be mediated by organic compounds of DEP.

Investigation of Vasodilatory Substances in Diesel Exhaust Particles (DEP): Isolation and Identification of Nitrophenol Derivatives

Vasodilatory compounds in the weak acidic fraction of a benzene extract of diesel exhaust particles (DEP) were fractionated by column chromatography through silica gel, and the chemical structures of these compounds were analyzed using GC-MS and 1H-NMR. The compounds in DEP that cause vasodilation — 3- and 4-nitrophenol, 2-methyl-4-nitrophenol, 3-methy-4-nitrophenol, and 4-nitro-3-phenylphenol — were isolated and identified. All five of these nitrophenols had vasodilatory activities (10-4 to 10-6 M) in rat thoracic artery assays, and 4-nitro-3-phenylphenol was the most potent vasodilator among these compounds. In addition, nine other alkylnitrophenols were isolated from the benzene extract of DEP and characterized.

Isolation of Nitrophenols from Diesel Exhaust Particles (DEP) as Vasodilatation Compounds

The compounds in diesel exhaust particles (DEP) that are responsible for vasodilatation were isolated and characterized for the first time. From benzene extract of DEP, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenl and 4-nitrophenol were isolated, and their vasodilatation activities were confirmed. 3-methyl-4-nitrophenol caused dilatation of rat thoracic artery, and the other two nitrophenols, also showed vasodilatation activities.

The cytotoxic effects of diesel exhaust particles on human pulmonary artery endothelial cells in vitro: role of active oxygen species

Diesel exhaust particles (DEP) have been proved to induce serious pulmonary injury, among which lethal pulmonary edema has been assumed to be mediated by vascular endothelial cell damage. In the present study, we investigated the cytotoxic mechanism of DEP on human pulmonary artery endothelial cells focusing on the role of active oxygen species. Endothelial cell viability was assessed by WST-8, a novel tetrazolium salt. Nitric oxide (NO) production was measured by using a new fluorescence indicator, diaminofluorescein-2 (DAF-2). Organic compounds in DEP were extracted by dichloromethane and methanol. DEP-extracts damaged endothelial cells under both subconfluent and confluent conditions. The DEP-extract-induced cytotoxicity was markedly reduced by treatment with SOD, catalase, N-(2-mercaptopropionyl)-glycine (MPG), or ebselen (a selenium-containing compound with glutathione peroxidase-like activity). Thus superoxide, hydrogen peroxide, and other oxygen-derived free radicals are likely to be implicated in DEP-extract-induced endothelial cell damage. Moreover, L-NAME and L-NMA, inhibitors of NO synthase, also attenuated DEP-extract-induced cytotoxicity, while sepiapterin, the precursor of tetrahydrobiopterin (BH 4, a NO synthase cofactor) interestingly enhanced DEP-extract-induced cell damage. These findings suggest that NO is also involved in DEP-extract-mediated cytotoxicity, which was confirmed by direct measurement of NO production. These active oxygen species, including peroxynitrite, may explain the mechanism of endothelial cell damage upon DEP exposure during the early stage.

Synergistic effect of diesel organic extracts and allergen Der p 1 on the release of chemokines by peripheral blood mononuclear cells from allergic subjects: involvement of the map kinase pathway.

The organic compounds of diesel exhaust particles (DEP-PAHs) have been shown to favor immunoglobulin production and bronchial hyperresponsiveness and to affect cytokine and chemokine productions. To evaluate if diesel exhaust could act in synergy with a house dust mite allergen (Der p 1), peripheral blood mononuclear cells from allergic patients were exposed to DEP-PAHs, with or without purified Der p 1. DEP-PAHs and Der p 1 separately induced an increase in interleukin (IL)-8, regulated on activation, normal T cells expressed and secreted (RANTES), and tumor necrosis factor-alpha concentrations. Interestingly, a synergy between the two stimuli was also observed. In the case of monocyte chemotactic protein (MCP)-1, DEP-PAHs reduced the release, whereas Der p 1 enhanced it. A simultaneous exposure led to reduced production as compared with allergen exposure alone, but still represented an increase as compared with the control exposure. Mitogen-activated protein (MAP) kinase Erk1/2 antagonist mainly inhibited the release of MCP-1, whereas MAP kinase p38 antagonist mainly suppressed the release of IL-8 and RANTES. Messenger RNA expression correlated with protein measurements. Moreover, supernatants from cells exposed to both DEP-PAHs and Der p 1 had a significant chemotactic activity on neutrophils and eosinophils. These findings suggest that simultaneous exposure of allergic patients to DEPs and allergens could result in high local chemokine levels via MAP kinase pathways activation, increasing the likelihood of reaching a critical threshold leading to the initiation of respiratory allergic symptoms.

Anti-estrogenic activity of diesel exhaust particles.

Estrogenic and anti-estrogenic activities of diesel exhaust particles (DEP) were evaluated using yeast cells expressing the human estrogen receptor and the responsive element regulating the expression of the receptor gene for beta-galactosidase (Routledge and Sumpter, 1996). It was found that a suspension of whole DEP suspension is not estrogenic but that this preparation possesses the ability to reduce the estrogen-dependent reporter activity. DEP were serially extracted with hexane, benzene, dichloromethane, methanol, and 1 M ammonia, and the estrogenic and anti-estrogenic activities of these preparations were determined. None of the extracts of DEP were estrogenic, but the extracts of benzene, dichloromethane and methanol possessed anti-estrogenic activity, and the activity of estrogen in the presence of hexane extract was slightly decreased. These results indicated that DEP contain heterologous compounds having anti-estrogenic activity. It is thought that those compounds in DEP can modulate the activity of estrogen, leading to the distruption of balance between estrogen and androgen. In this paper, the environmental effects of DEP in relation to the endocrine disrupting effect of organic compounds in DEP are discussed.

460 Synergistic effects of diesel organic extracts and allergen Der p 1 on the release of chemokines by peripheral blood mononuclear cells from allergic subjects

The organic compounds of diesel exhaust particles (DEP-PAHs) have been shown to favor immunoglobulin production and bronchial hyperresponsiveness and to affect cytokine and chemokine productions. To evaluate if diesel exhaust could act in synergy with a house dust mite allergen (Der p 1), peripheral blood mononuclear cells from allergic patients were exposed to DEP-PAHs, with or without purified Der p 1. DEP-PAHs and Der p 1 separately induced an increase in interleukin (IL)-8, regulated on activation, normal T cells expressed and secreted (RANTES), and tumor necrosis factora concentrations. Interestingly, a synergy between the two stimuli was also observed. In the case of monocyte chemotactic protein (MCP)-1, DEPPAHs reduced the release, whereas Der p 1 enhanced it. A simultaneous exposure led to reduced production as compared with allergen exposure alone, but still represented an increase as compared with the control exposure. Mitogen-activated protein (MAP) kinase Erk1/2 antagonist mainly inhibited the release of MCP-1, whereas MAP kinase p38 antagonist mainly suppressed the release of IL-8 and RANTES. Messenger RNA expression correlated with protein measurements. Moreover, supernatants from cells exposed to both DEP-PAHs and Der p 1 had a significant chemotactic activity on neutrophils and eosinophils. These findings suggest that simultaneous exposure of allergic patients to DEPs and allergens could result in high local chemokine levels via MAP kinase pathways activation, increasing the likelihood of reaching a critical threshold leading to the initiation of respiratory allergic symptoms.