Takamichi Ichinose

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.

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.

Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and pentane in respired gases of rats

Abstract This research measured lipid peroxidation products in rats of varying age by a new method. Wistar male rats, 4, 12, 22 and 32 months old, were examined for lipid peroxidation in vivo by measurement of ethane, ethylene, butane and pentane in breath gases of intact animals. In the older rats, the amounts of the four hydrocarbons exhaled were greater than those in younger rats. All hydrocarbons tested were related to age by an exponential relationship, and quantitatively, ethane and ethylene were related to age with a linear regression fit correlation coefficient, 0.466–0.622 (p ethane>butane. There were significant differences in the production of all hydrocarbons between 32 month-old rats and 4 month-old rats. Thiobarbituric acid reactants in serum exhibited an increasing tendency with age, but the values of 32 month-old rats were lower than those of 22 month-old rats. However, the differences between the age groups were not significant. These results showed that the measurement of hydrocarbons in the ratsbreath was a sensitive index of in vivo peroxidation during aging.

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.

IL-4 production in mediastinal lymph node cells in mice intratracheally instilled with diesel exhaust particulates and antigen

To clarify the relationship between air pollutants and IgE antibody production, interleukin 4 (IL-4) production was investigated in BALB/c mice intratracheally injected with diesel exhaust particulates (DEP) mixed with antigen (Ovalbumin (OA) or Japanese Cedar Pollen (JCP)). BALB/c mice were injected with DEP plus OA or OA alone three times with a 3-week interval. After the last instillation, proliferative response and lymphokine-producing activity of mediastinal lymph node cells (LNC) were examined in vitro. Proliferative response to OA in mediastinal LNC from mice injected with DEP plus OA was enhanced 4-17 times of that from control mice. IL-4-producing activity by OA stimulation also enhanced in mediastinal LNC from mice injected with DEP plus OA. A significantly larger amount of anti-OA IgE antibody was detected in sera from DEP- and OA-injected mice compared with those from control mice. The levels of IL-4, estimated by JCP antigen in mediastinal LNC, from mice injected with DEP plus JCP were two-fold higher than those from mice injected with JCP alone. These results suggest that intratracheal instillation of DEP affects antigen-specific IgE antibody responses via local T-cell activation, especially enhanced IL-4 production.

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.

Studies on biochemical effects of nitrogen dioxide. II. Changes of the protective systems in rat lungs and of lipid peroxidation by acute exposure

This work was done to clarify the relation between the change of lipid peroxidation and the protective systems in lungs after NO2 exposures. JCL:Wistar 8-wk-old male rats were exposed continuously to 10 ppm NO2 for 2 wk. Lipid peroxidation, measured by ethane exhalation in the breath of the rats and by the reaction of thiobarbituric acid with lung homogenates, increased to a maximum at 3 d after a decline at 1 d, and then returned to the initial level (of d 0). Activities of glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), disulfide reductase (DSR), and superoxide dismutase (SOD) in the 105,000 X g supernatant of lung homogenates were depressed slightly at 1 d. Thereafter, they increased significantly to their maximum levels from 5 to 10 d, and these maximum levels were maintained until d 14. The pattern of change of these protective enzymes was symmetric to that of lipid peroxidation after 3 d. The order of the ratio of the increased value to the initial value was G6PD greater than DSR greater than 6PGD greater than GR greater than GPx greater than SOD. The time course of nonprotein sulfhydryls was similar to that of the protective enzymes. In contrast, the amounts of vitamin E increased to a maximum at 2 d and then returned to the initial level. The periodic change of vitamin E was similar to that of lipid peroxidation rather than that of the protective enzymes. These results suggest that the ability of the enzyme systems in lungs to protect against NO2 fluctuated in a complex manner and the activities of the protective enzymes varied inversely with lipid peroxidation.

Diesel Exhaust Inhalation Enhances Airway Hyperresponsiveness in Mice

Background: Repeated intratracheal instillation of diesel exhaust particles and ovalbumin-induced airway hyperresponsiveness and airway inflammation in mice. However, the effects of daily inhalation of diesel exhaust may differ from the effects of direct instillation. Methods: Therefore, mice were exposed to diesel exhaust by inhalation 12 h per day for 3 months. Before the diesel exhaust exposure, ovalbumin was injected intraperitoneally as a sensitization. After 3 weeks of diesel exhaust exposure, these mice were challenged with ovalbumin every 3 week thereafter. Results: Diesel exhaust exposure with antigen challenge induced airway hyperresponsiveness and airway inflammation which was characterized by increased numbers of eosinophils and mast cells in lung tissue. The recruitment of inflammatory cells was accompanied by an increment in goblet cells on bronchial epithelium. Diesel exhaust exposure alone also enhanced airway hyperresponsiveness, but did not induce eosinophilic infiltration and/or an increment in goblet cells. Conclusion: Diesel exhaust inhalation enhanced airway hyperresponsiveness and airway inflammation caused by ovalbumin sensitization in mice.