Exposure to diesel exhaust (DE) is known to cause lung tumors in rats. To clarify the mutagenicity of DE, we estimated mutant frequency (MF) and determined the mutation spectra in rat lung after ...exposure to DE using lambda/lacI transgenic rats (Big Blue® system). Male Big Blue® rats (6 weeks old) were exposed for 4 weeks to 1 or 6 mg/m3 DE, which contains suspended particulate matter. Control rats were maintained in filtered clean air. After exposure to 6 mg/m3 DE, MF in lung was 4.8-fold higher than in control rats (P < 0.01), but no increase in MF was observed in rats exposed to 1 mg/m3 DE. Sixty-nine mutants were identified after exposure to 6 mg/m3 DE. The major mutations were A:T→G:C (18 mutations) and G:C→A:T (19 mutations) transitions. Remarkably, G→T transversion of the lacI gene at site 221 was a hot-spot induced by exposure to DE, and there were complex mutations in which multiple mutations occurred in a single mutant, especially in the rats exposed to 6 mg/m3 DE. DNA adducts formed by DE were analyzed using a 32P-post-label TLC method and the amount of 8-hydroxydeoxyguanosine (8-OHdG) was measured using HPLC. Relative adduct level and amount of 8-OHdG were significantly increased in the rats exposed to 6 mg/m3 DE compared with the controls (3.0- and 2.2-fold, respectively; P < 0.01). The level of cytochrome P450 1A1 mRNA was shown by northern blot analysis to be significantly increased in the lungs of rats exposed to 6 mg/m3 DE (5.5-fold; P < 0.01). These results indicate that DE causes lesions in genomic DNA and acts as a mutagen in rat lung.
Epidemiologic and experimental studies suggest that diesel exhaust particles (DEPs) may be related to increasing respiratory mortality and morbidity. We have shown that DEPs augmented the production ...of inflammatory cytokines by human airway epithelial cells in vitro. To better understand the mechanisms of their proinflammatory activities, we studied the effects of several components extracted from DEPs on interleukin (IL)-8 expression in human bronchial epithelial cell line BEAS-2B and normal human airway epithelial cells obtained from very peripheral airways by an ultrathin bronchoscope. We used several agents active on signal transduction pathways in cytokine expression, such as the protein kinase C inhibitor staurosporin, antioxidant agents including N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Benzene-extracted components showed effects mimicking DEPs on IL-8 gene expression, release of several cytokines (IL-8; granulocyte macrophage colony-stimulating factor; and regulated on activation, normal T cells expressed and secreted) and nuclear factor (NF)-kappa B activation. We also found that NAC, PDTC, and SB203580 suppressed the activities of DEPs and their benzene extracts, suggesting the roles of oxidants-mediated NF-kappa B activation and p38MAPK pathways. Finally, benzoapyrene, one of the important compounds included in the benzene component, replicated the activities shown by DEPs.
To elucidate whether immunoglobulin (Ig) E or IgG are involved in the murine asthma model, we compared the pathogenic features of mice that were high IgG responders (C3H/He) with mice that were high ...IgE responders (BALB/c) after intratracheal instillation of diesel exhaust particles (DEP) and ovalbumin sensitization. Both mouse strains received DEP intratracheally once a week for 5 weeks. After the second injection of DEP, ovalbumin and aluminium hydroxide were injected intraperitoneally. After the last DEP administration, the mice were challenged by exposure to an aerosol of ovalbumin. DEP caused increased IgG1 production and airway hyperresponsiveness after ovalbumin sensitization in C3H/He mice, although IgE production did not change in either strain. Furthermore, in C3H/He mice, the number of eosinophils and goblet cells in the bronchial epithelium, and the expression of interleukin-5 and interleukin-2 were increased by DEP and ovalbumin treatments. In contrast, the pathogenic changes in BALB/c mice were weak, even though the same protocol was used. In conclusion, murine strain differences in response to air pollutants and allergens seem to be related to antigen-specific immunoglobulin G1 production and cytokine expression in the lungs.
Previous experimental studies have suggested that nasal instillation of diesel exhaust particles (DEP) can enhance nasal IgE response and cytokine production. However, there is no experimental ...evidence for the relation of DEP to allergic asthma. We investigated the effects of DEP inoculated intratracheally on antigen-induced airway inflammation, local expression of cytokine proteins, and antigen-specific immunoglobulin production in mice. DEP aggravated ovalbumin-induced airway inflammation characterized by infiltration of eosinophils and lymphocytes and an increase in goblet cells in bronchial epithelium. DEP with antigen markedly increased interleukin-5 (IL-5) protein levels in lung tissue and bronchoalveolar lavage supernatants compared with either antigen or DEP alone. The combination of DEP and antigen induced significant increases in local expression of IL-4, granulocyte macrophage-colony stimulating factor (GM-CSF), and IL-2, whereas expression of interferon-gamma was not affected. In addition, DEP exhibited adjuvant activity for the antigen-specific production of IgG and IgE. These results provide the first experimental evidence that DEP can enhance the manifestations of allergic asthma. The enhancement may be mediated mainly by the increased local expression of IL-5, and also by the modulated expression of IL-4, GM-CSF, and IL-2.
Abstract
We have previously reported that intratracheal instillation of diesel exhaust particles (DEP) enhances allergen-induced eosinophilic airway inflammation, local expression of interleukin-5 ...and granulocyte macrophage-colony stimulating factor, and allergen-specific production of IgE and IgG in mice. The present study was undertaken to elucidate the effects of DEP on airway hyperresponsiveness as another characteristic feature of allergic asthma. The animals were randomized into four experimental groups that received intratracheal instillation with vehicle, ovalbumin (OVA), DEP, or the combination of OVA and DEP on a weekly basis for 6 weeks. Respiratory resistance (Rrs) was measured 24 h after the last instillation. An increase in Rrs in animals that inhaled acetylcholine was significantly greater in the combined treatment with OVA and DEP than in the other treatments. The present study indicates that DEP can enhance airway responsiveness associated with allergen exposure, and provides experimental evidence that DEP may deteriorate the pathophysiology of allergen-related respiratory disease such as allergic asthma.
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.
Diesel exhaust particles (DEPs) contain polycyclic aromatic hydrocarbons (PAH) that bind to aryl hydrocarbon receptors (AhRs) and decrease sperm production. Since it is not clear if AhR mediates DEP ...toxicity, we investigated the effect of DEPs in four strains of mice that have different AhR responsiveness. We treated BALB/c, C57BL/6, ICR and DBA/2 mice with DEP suspensions and compared their toxicity in each strain. In both the vehicle- and DEP-treated groups, ethoxyresorufin-O-deethylase (EROD) activity, as an indirect index of AhR activity, was increased in the order of BALB/c C57BL/6 ICR DBA/2. Only BALB/c and C57BL/6 mice had significantly lower daily sperm production (DSP) than vehicle-treated mice. All strains exhibited increased sperm abnormalities. In particular, the C57BL/6, ICR and DBA/2 mice exhibited significantly increased abnormalities. A significant correlation was found between EROD activity and DSP or incidence of morphologically abnormal sperm. These data suggest that DEP toxicity may affect the male reproductive system in an AhR-dependent manner.
Chronic airway inflammation, mucus hypersecretion, reversible airway constriction, and bronchial hyperresponsiveness are important pathogenic features of asthma. We found that diesel exhaust ...particles (DEP) instilled intratracheally and repeatedly to mice (once/week for 16 weeks) caused marked infiltration of inflammatory cells, proliferation of goblet cells, increased mucus secretion, respiratory resistance, and airway constriction. Eosinophils in the submucosa of the proximal bronchi and medium bronchioles increased eightfold following instillation. Eosinophil infiltration was significantly suppressed by pretreatment with polyethyleneglycol-conjugated superoxide dismutase (PEG-SOD). Bound sialic acid concentrations in bronchial alveolar lavage fluids, an index of mucus secretion, increased with DEP, but were suppressed by pretreatment with PEG-SOD. Goblet cell hyperplasia, airway narrowing, and airway constriction also were observed with DEP. Respiratory resistance in the DEP-group to acetylcholine was 11 times higher than in controls, and the increased resistance was significantly suppressed by PEG-SOD pretreatment. These findings suggest that DEP and/or oxygen radicals derived from DEP cause bronchial asthma in mice.
In an experiment to clarify the involvement of oxygen radicals in lung carcinogenesis induced by diesel exhaust particles (DEP), we found that there is a strong relation between lung tumor response ...and formation of 8-hydroxydeoxyguanosine (8-OHdG) in lung DNA of mice administered DEP by repeated intratracheal instillation. Repeated intratracheal instillation of DEP also induced the activity of cytochrome P-450 reductase in the lungs as a representative enzyme of superoxide generation, and two types of nitric oxide (NO) synthase, cNOS and iNOS, in the lungs. On the other hand, activities of CuZn-superoxide dismutase (SOD) and Mn-SOD antioxidant enzymes were depressed by the instillation of DEP. These results suggest that generation of superoxide, hydroxyI radical, and nitric oxide are increased in epithelial cells in airways, and that the increased superoxide and nitric oxide react very easily to produce peroxynitrite (ONOO
−
). The peroxynitrite also produce hydroxyI radical. The hydroxyl radical may play an important role in carcinogenesis by DEP.
This study was undertaken to investigate the effect of azuki bean (
Vigna angularis) seed coats (ABSC), which contain polyphenols, on the infiltration of macrophages and the progression of diabetic ...nephropathy in streptozotocin (STZ)-induced diabetic rats. The diabetic rats were divided into three groups with 0% (commercial diet), 0.1% and 1.0% ABSC diets. The vehicle-injected controls were given a commercial diet. At 10 weeks, the macrophage kinetics, the degree of fibrosis in glomeruli and mRNA expression for monocyte chemoattractant protein-1 (MCP-1) were examined. There was no difference in plasma glucose levels between diabetic rats treated with and without ABSC. The plasma levels of malondialdehyde (MDA) in the ABSC-treated diabetic rats were significantly lower than those in the untreated diabetic rats. Histopathologically, the percentage of the fibrotic areas stained by Sirius red stain in the glomeruli in the ABSC-treated diabetic rats was lower than in the untreated diabetic rats. ED1-positive macrophages in the glomeruli and tubulointerstitium in the untreated diabetic rats showed a significant increase in number compared with the controls. In contrast, the number of macrophages in the ABSC-treated diabetic rats was smaller than that in untreated diabetic rats. MCP-1 mRNA expression, estimated by real-time quantitative RT-PCR, was increased 2.5-fold in the untreated diabetic rat kidney, while a lower level was observed in the ABSC-treated diabetic rats. In conclusion, our results suggest that ABSC treatments suppress the increased number of infiltrating macrophages and MCP-1 mRNA expression, and attenuated the glomerular expansion in STZ-induced rat diabetic nephropathy.
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