Masaru Sagai

Biological effects of diesel exhaust particles. I. In vitro production of superoxide and in vivo toxicity in mouse.

The problem of whether or not active oxygen species are involved in pulmonary injury by diesel exhaust particles (DEP) was investigated. We found that DEP could produce superoxide O2.- and hydroxyl radical (.OH) in vitro without any biological activating systems. In this reaction system, O2.- and .OH productions were inhibited by addition of superoxide dismutase (SOD) and dimethylsulfoxide, respectively. DEP which were washed with methanol could no longer produce O2.- and .OH, indicating that active components were extractable with organic solvents. These oxygen radicals were also identified by electron spin resonance (ESR) measurement. Furthermore, DEP instilled intratracheally to mouse caused high mortality at low dose, although methanol-washed DEP did not kill any mouse. The cause of death seemed to be pulmonary edema mediated by endothelial cell damage. The instilled DEP markedly decreased the activities of SOD, glutathione peroxidase, and glutathione S-transferase in mouse lungs. On the other hand, the death rate and lung injury were markedly prevented by polyethylene glycol conjugated SOD (PEG-SOD) pretreatment prior to DEP administration. The mortality and lung injury by DEP were also suppressed by butylated hydroxytoluene (BHT) pretreatment. From these results, it was suggested that most parts of DEP toxicity in lungs are due to active oxygen radicals such as O2.- and .OH, and that the cause of death is due to pulmonary edema mediated by endothelial cell damage.

Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage.

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 micrograms) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide.

Biological effects of diesel exhaust particles (DEP). III Pathogenesis of asthma like symptoms in mice

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.

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.

Biological effects of diesel exhaust particles (DEP). II. Acute toxicity of DEP introduced into lung by intratracheal instillation

Histopathological examination and cytological analyses in bronchial alveolar lavage fluids (BALF) were performed to clarify the acute toxicity of diesel exhaust particles (DEP) introduced into the lung of ICR mice by intratracheal instillation. Activated charcoal (Norit) was intratracheally administered as a control for non-oedemagenic carbon particles. After administration of two doses (0.4 mg or 0.8 mg per mouse) of DEP, lung water contents increased with instillation dose and with time and increased 1.9 and 2.7-fold, respectively, compared to control animals 24 h after the administration of DEP. In contrast, the instillation of Norit had no effect on the increase in water contents. An inflammatory response in lungs was observed by an increase of inflammatory cells in BALF from mice instilled with DEP. The degree of increase in neutrophils of BALF from mice treated with DEP was much greater than in mice treated with Norit. An intense color of MB-pigment, which showed the extent and degree of endothelial cell injury, was found up to 4 h after administration of DEP. Histopathologically, the disruption of capillary endothelial cells, the detachment from their basement membrane and necrosis, disruption and desquamation of type I pneumocytes were observed, 6 h after the injection of DEP, by electron microscopy. An influx of neutrophils into alveoli, intra-alveolar hemorrhage, perivascular oedema and bronchiolar cell hypertrophy were detected between 18 and 24 h after DEP administration. However, the magnitude of these appearances was greater in mice treated with 0.8 mg of DEP than in mice treated with 0.4 mg. The administration of Norit caused an increase of alveolar macrophages and slight infiltration of neutrophils into the alveolar air spaces and alveolar septa in the animals and had no effects on the bronchioles. These results may suggest that damage of capillary endothelial cells and type I pneumocytes are the earliest changes of lung toxicities by DEP and these cell injuries lead to alveolar oedema and the subsequent inflammatory response.

Involvement of superoxide and nitric oxide on airway inflammation and hyperresponsiveness induced by diesel exhaust particles in mice

We previously demonstrated that chronic intratracheal instillation of diesel exhaust particles (DEP) induces airway inflammation and hyperresponsiveness in the mouse, and that these effects were partially reversed by the administration of superoxide dismutase (SOD). In the present study, we have investigated the involvement of superoxide in DEP-induced airway response by analyzing the localization and activity of two enzymes: (1) a superoxide producer, NADPH cytochrome P-450 reductase (P-450 reductase), and (2) a superoxide scavenger, SOD, in the lungs of the exposed mice and controls. P-450 reductase was detected mainly in ciliated cells and clara cells: its activity was increased by the repeated intratracheal instillation of DEP. While CuZn-SOD and Mn-SOD were also present in the airway epithelium, their activity was significantly decreased following DEP instillation. Exposure to DEP doubled the level of nitric oxide (NO) in the exhaled air. DEP exposure also increased the level of constitutive NO synthase (cNOS) in the airway epithelium and inducible NO synthase (iNOS) in the macrophages. Pretreatment with N-G-monomethyl L-arginine, a nonspecific inhibitor of NO synthase, significantly reduced the airway hyperresponsiveness induced by DEP. These results indicate that superoxide and NO may each contribute to the airway inflammation and hyperresponsiveness induced by the repeated intratracheal instillation of DEP in mice.

Comparison of ciliary activity and inflammatory mediator release from bronchial epithelial cells of nonatopic nonasthmatic subjects and atopic asthmatic patients and the effect of diesel exhaust particles in vitro.

BACKGROUND Recent studies have suggested that asthmatic patients may be more susceptible to the adverse effects of air pollutants, including diesel exhaust particles (DEP). The underlying mechanisms, however, are not clear. METHODS We cultured bronchial epithelial cells from bronchial biopsy specimens of well-characterized groups of nonatopic, nonasthmatic individuals and atopic patients with mild asthma and compared the ciliary beat frequency (CBF) and release of IL-8, GM-CSF, regulated on activation, normal T-cell expressed and secreted (RANTES), and soluble intercellular adhesion molecule-1 (sICAM-1) from these cells both before and after exposure for 24 hours to 10 to 100 micrograms/mL DEP in vitro. RESULTS The baseline CBF was not found to be significantly different in the bronchial epithelial cell cultures of nonasthmatic and asthmatic individuals. Incubation with DEP significantly attenuated the CBF of both the nonasthmatic and asthmatic bronchial epithelial cell cultures at all concentrations of DEP investigated and were maximal (55.5% and 45.2%, respectively) after incubation with 100 micrograms/mL DEP. The bronchial epithelial cell cultures of asthmatic patients constitutively released significantly greater amounts of IL-8, GM-CSF, and sICAM-1 than bronchial epithelial cell cultures of nonasthmatic subjects. The cultures of only asthmatic patients additionally released RANTES. Incubation of the asthmatic cultures with 10 micrograms/mL DEP significantly increased the release of IL-8 (from 102.0 to 167.8 pg/micrograms cellular protein; P <.01), GM-CSF (from 0.43 to 1.87 pg/micrograms cellular protein; P <.01), and sICAM-1 (from 14.7 to 38.1 pg/micrograms cellular protein; P <.02) after 24 hours. Incubation with 50 to 100 micrograms/mL DEP, however, significantly decreased the release of IL-8 and RANTES from these cultures. In contrast, only the higher concentrations of 50 to 100 micrograms/mL DEP significantly increased release of IL-8 (from 37.9 to 71.5 pg/micrograms cellular protein; P <.05) and GM-CSF (from 0.06 to 0. 34 pg/micrograms cellular protein; P <.05) from the bronchial epithelial cells of nonasthmatic individuals. CONCLUSIONS These results suggest that bronchial epithelial cells of asthmatic subjects are different from bronchial epithelial cells of nonasthmatic subjects with regard to the amounts and types of proinflammatory mediators they can release and that the increased sensitivity of bronchial epithelial cells of asthmatic subjects to DEP may possibly result in exacerbation of their disease symptoms.

Effects of diesel exhaust on allergic airway inflammation in mice.

BACKGROUND Eosinophilic infiltration and goblet cell hyperplasia were induced by the intratracheal instillation of diesel exhaust particles and ovalbumin in mice. However, it is unknown whether its results differ from the effects of the inhalation of diesel exhaust and allergen. OBJECTIVES The purpose of this study was to compare the effects of diesel exhaust inhalation and intratracheal instillation of diesel exhaust particles in a murine asthma model. METHODS ICR mice were exposed to 3 mg soot per cubic meter of diesel exhaust for 6 weeks. After the first week, animals were sensitized by intraperitoneal injection of ovalbumin and aluminum hydroxide gel. After 5 weeks of diesel exhaust exposure, the mice were challenged with ovalbumin. The animals were killed 1, 2, 3, and 7 days after the challenge and investigated for airway inflammation, hyperplasia of goblet cells, airway hyperresponsiveness, local cytokine expression, and antigen-specific IgE and IgG1 production. RESULTS Exposure to diesel exhaust enhanced infiltration of eosinophils and neutrophils in murine airways even 1 day after the challenge. An increment of goblet cells under the bronchial epithelium was followed by the recruitment of inflammatory cells. Furthermore, exposure to diesel exhaust combined with ovalbumin sensitization enhanced respiratory resistance and expression of IL-5 in lung tissue and IgG1 production but not IgE. However, diesel exhaust alone did not induce pathologic changes in mice. CONCLUSIONS Diesel exhaust enhanced allergic airway inflammation, hyperplasia of goblet cells, and airway hyperresponsiveness caused by ovalbumin sensitization.

Murine strain differences in allergic airway inflammation and immunoglobulin production by a combination of antigen and diesel exhaust particles.

To clarify the relationship between the manifestations of allergic airway inflammation modulated by diesel exhaust particles (DEP) and immunoglobulin production in response to an antigen, airway inflammation characterized by the infiltration of eosinophils, goblet cell proliferation, and antigen-specific immunoglobulin (Ig) production was investigated in five strains of mice after immunization with ovalbumin (OA). Mice were injected intratracheally with OA (1 microg) or OA (1 microg) + DEP (50 microg) four times at 3-week intervals. The order of antigen-specific IgG1 production in plasma of mouse strains treated with OA alone was CBA/2N <BDF/1 <C57BL/6 < ICR <C3H/He. The adjuvant effect of DEP on IgG1 production was observed in CBA/2N, BDF/1, ICR, and C57BL/6 mice. The immune activity in BDF/1 mice was significantly elevated (P < 0.01). The OA-specific IgE in plasma was unaffected by antigen challenge with or without DEP in any strain. The degree of eosinophilic inflammation and goblet cell proliferation in the airway induced by OA alone or OA + DEP corresponded well with the antigen-specific IgG1 production. These results suggest that the manifestations of allergic airway inflammation modulated by DEP were closely related to the immunoglobulin production response to OA, especially with regard to enhanced IgG1 production.

Diesel Exhaust Particles Enhance Airway Responsiveness Following Allergen Exposure in Mice

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.