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.

Apparent inhibition of superoxide dismutase activity in vitro by diesel exhaust particles.

The inhibitory effects of diesel exhaust particles (DEP) on superoxide dismutase (SOD) activity were examined in vitro because intratracheal administration of DEP to mice resulted in a suppression of the pulmonary enzyme activity (Sagai et al., Free Radic. Biol. Med. 14:37-47; 1993). Superoxide production, based on the reduction of cytochrome c, was suppressed considerably by the soluble fraction of mouse lung and by purified SOD from bovine erythrocytes, but the suppression was drastically diminished in the presence of methanol-extractable compounds of DEP. Inhibition of SOD by diethyldithiocarbamate was irreversible, but that by 1,2-naphthoquinone (1,2-NQ) and the methanol extract of DEP was removed by dialysis. Inhibition of superoxide mediated cytochrome c reduction by Tiron, a scavenging agent for superoxide, was blocked by the methanol extract and 1,2-NQ in a concentration-dependent manner. In contrast, addition of a large amount of SOD to the reaction mixture resulted in an almost complete disappearance of inhibitory action of not only 1,2-NQ but also the methanol extract. The existence of carbonyl compounds in the DEP was confirmed by thin-layer chromatography (TLC) with 2,4-dinitrophenylhydrazine reagent. Electron spin resonance (ESR) spectra of an incubation mixture of oxidized 1,2-dihydroxynaphthalene in the absence and presence of cytochrome c indicated a reaction between the semiquinone radical of 1,2-NQ and cytochrome c. These results indicate that the apparent reduction in SOD activity by DEP is due to the chemical reaction of superoxide with components like quinones, which reduce levels of superoxide.

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.

8-Hydroxyguanosine formed in human lung tissues and the association with diesel exhaust particles.

Diesel exhaust particles consist of various organic chemicals, heavy metals, and carbon particles. Knowledge of the fate of organic chemicals and carbon particles in the lungs is important to determine the mechanisms responsible for lung tumors. In the present study, diesel particle extracts were found to show mutagenicity for YG3003, a sensitive strain to some oxidative mutagens, as well as other mutant strains, and those of lung tissues obtained from lung cancer patients exhibited potent mutagenicity. Formation of 8-hydroxyguanosine (8-OHdG) as a biomarker of oxidative damage was analyzed with in vitro and in vivo assay systems. The 8-OHdG was detected in all 22 cases of lung tissues with carcinomas tested and their levels increased with the increasing age of the patients, suggesting a correlation between age and the presence of carbon particles in lung tissues. Therefore, the formation of 8-OHdG due to diesel exhaust particles was investigated via intratracheal injections into mice. 8-OHdG formation was elevated when carboneceous particles, after removal of organic chemicals with various solvents, were administered to mice, but it was not elevated when polyaromatic compounds such as benzo[a]pyrene, 1,8-dinitropyrene, and 1-nitropyrene were used in the same procedure in mice. The carboneceous particles were formed from a giant particle that was aggregated by micro-particles with diameters of 1.47 +/- 1.34 to 1.05 +/- 0.83 microm. These results suggest that carboneceous particles, but not mutagens and carcinogens, promote the formation of 8-OHdG, and that as a mechanism, alveolar macrophages may be involved in oxidative damage. The oxidative damage may be due to the fact that the mutation is involved with the generation of a hydroxyl radical during phagocytosis, and the hydroxyl radical leads to hydroxylation at the C-8 position of the deoxyguanosine residue in the DNA.

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.

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.

Serum antioxidant vitamins and risk of lung and stomach cancers in Shenyang, China.

In a hospital-based case control study, we measured serum concentrations of vitamin A, beta-carotene and vitamin E for subjects with cancer (58 cases of lung cancer and 22 cases of stomach cancer) and 63 matched controls in Shenyang, China. Lung cancer patients had significantly (P < 0.01) lower mean serum levels of vitamin A, beta-carotene and vitamin E than controls, while the mean serum level of vitamin E did not differ between stomach cancer patients and the controls. Lower serum levels of vitamin A, vitamin E and beta-carotene were associated with an increased risk of lung cancer. Lower serum levels of vitamin A and beta-carotene were associated with a higher risk of stomach cancer, although the number of cases was small. An increased risk of lung cancer associated with lower serum levels of vitamin A and vitamin E was more evident among heavy smokers than among non-heavy smokers.

Lipid peroxidation and antioxidative protection mechanism in rat lungs upon acute and chronic exposure to nitrogen dioxide.

This work was done to clarify the relation between the changes of lipid peroxidation and the activities of antioxidative protective enzymes in lungs of rats exposed acutely, subacutely, and chronically to nitrogen dioxide. It was confirmed that the activities of the antioxidative enzymes to protect cells from oxidative stress increased in an early phase, and then the activities decreased gradually. Lipid peroxides increased once in an early phase and then returned to the control level; thereafter, lipid peroxides increased gradually again. Lipid peroxidation as measured by ethane exhalation increased significantly with 0.04, 0.4, and 4 ppm nitrogen dioxide exposure for 9, 18, and 27 months, and a dose-response relationship was clearly observed. The temporal changes of lipid peroxidation varied inversely with that of the activities of antioxidative protective enzymes. From these results, it was suggested that the increments of antioxidative protective enzyme activities in an early phase were complementary effects to protect cells from damage by lipid peroxides which were increased by nitrogen dioxide exposure, and that the complementary effects are lost in later phases of life-span exposure. Finally, loss of such protective complementary effects might relate to some chronic diseases in lungs. Therefore, the temporal changes described above are important characteristics in chronic exposure of air pollutants.

Oxygen radicals in lung carcinogenesis accompanying phagocytosis of diesel exhaust particles.

We sought to examine the involvement of oxygen radicals derived from phagocytosis process in lung carcinogenesis induced by diesel exhaust particles (DEP). The carcinogenic response and formation of 8-hydroxydeoxyguanosine (8-OHdG) were examined in the lungs of mice intratracheally injected with washed DEP (WDEP), DEP, or nontoxic control particles of titanium dioxide (TiO2). After 10 weekly treatments with these particles, the formation of 8-OHdG in the lungs of mice treated with WDEP or DEP showed a significant increase, but not in those treated with TiO2. After 12 months, the incidence of lung tumors in mice treated with WDEP or DEP was higher than that of mice treated with vehicle by 2.3- and 3.1-fold, respectively. A significant difference in the incidence of tumors was found between the vehicle group and DEP-treated group. Treatment with TiO2 had no effect on the incidence of lung tumors. The formation of 8-OHdG in mice treated with these particles was significantly correlated with the development of lung tumors. These results suggest that the induction of DNA damage by oxygen radicals may be an important factor in the initiation of WDEP- and DEP-induced lung carcinogenesis, and that oxygen radicals derived from the phagocytic process may play a role in 8-OHdG formation induced by DEP.

Long-term exposure to diesel exhaust enhances antigen-induced eosinophilic inflammation and epithelial damage in the murine airway.

The histopathologic changes in the murine airway induced by long-term exposure to diesel exhaust (DE), ovalbumin (OA), or both were investigated. The relationship between the histopathologic appearances in the airway and immunoglobulin production or local cytokine levels in the lungs was also studied. ICR mice were exposed to clean air or DE at a soot concentrations of 0.3, 1.0, or 3.0 mg/m3 for 34 weeks. Fifteen weeks after exposure to DE, mice were sensitized intraperitoneally with 10 micrograms of OA and challenged by an aerosol of 1% OA six times at 3-week intervals during the last 18 weeks of the exposure. DE exposure caused a dose-dependent increase of nonciliated cell proliferation and epithelial cell hypertrophy in the airway, but showed no effect on goblet cell proliferation in the bronchial epithelium and eosinophil recruitment in the submucosa of the airway. OA treatment induced very slight changes in goblet cell proliferation and eosinophil recruitment. The combination of OA and DE exposure produced dose-dependent increases of goblet cells and eosinophils, in addition to further increases of the typical changes induced by DE. OA treatment induced OA-specific IgG1 and IgE production in plasma, whereas the adjuvant effects of DE exposure on immunoglobulin production were not observed. Inhalation of DE led to increased levels of IL-5 protein in the lung at a soot concentration of 1.0 and 3.0 mg/m3 with OA, although these increases did not reach statistical significance. We conclude that the combination of antigen and chronic exposure to DE produces increased eosinophilic inflammation, and cell damage to the epithelium may depend on the degree of eosinophilic inflammation in the airway.

Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury.

Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg-1 day-1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1. 6-1.9-fold, 5-8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000xg supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11-43 microM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.

Biochemical effects of combined gases of nitrogen dioxide and ozone. III. Synergistic effects on lipid peroxidation and antioxidative protective systems in the lungs of rats and guinea pigs.

Rats and guinea pigs were exposed continuously to 0.4 ppm NO2, 0.4 ppm O3 or a combination of the two gases for 2 weeks. The concentration of lipid peroxides in lungs of rats and guinea pigs exposed to NO2 alone or O3 alone did not change. The lipid peroxide level of rats inhaling the combined gases also did not change. However, the level of lipid peroxides in guinea pigs exposed to a combination of the two gases was increased to 2.2 times of the control level, showing a synergistic interaction. No increases of antioxidative protective enzyme activities and of antioxidants (such as NPSH, VE, VC) in guinea pigs exposed to NO2, O3 or the combined gases were found. In rats, no changes in enzyme activities and of the antioxidant contents were observed after NO2 alone, but O3 exposure produced slight increases of NPSH, VC, and GPx-H2O2. On the other hand, in rats exposed to the combined gases, marked synergistic increased of many antioxidative factors such as NPSH, VC, G6PD, GPx-cum.OOH and GPx-H2O2 were found. The results show that those animals which are able to increase antioxidative protective factors in the lung following exposure to the combined gases do not respond with a significant increase in lipid peroxides. On the other hand, in animals with poor induction-ability of these factors lipid peroxides are formed. This might explain why guinea pigs were the most sensitive to the effects of the combined gases. Furthermore, it was shown that in guinea pigs the increased level of lipid peroxides and that in rats the increased activities of antioxidative enzymes and the increased contents of the antioxidants were synergistic following exposure to the combined gases.

Combined exposure to NO2, O3 and H2SO4-aerosol and lung tumor formation in rats.

The promoting effects of a combined exposure to two pollutants (NO2, O3 or H2SO4-aerosol) at near ambient levels on lung tumorigenesis induced by N-bis(2-hydroxypropyl) nitrosamine (BHPN) were investigated in male Wistar rats. The rats were given a single intraperitoneal injection of BHPN (0.5 g per kg body wt.) at 6 weeks of age. They then were exposed to clean air, 0.05 ppm O3 (mean concentration for 10 h/day; 0.1 ppm peak concentration), 0.05 ppm O3 (mean concentration for 10 h/day; 0.1 ppm peak concentration) + 0.4 ppm NO2 or 0.4 ppm NO2 + 1 mg/m3 of H2SO4-aerosol for 13 months and were then maintained in a clean room for another 11 months. Room control animals were kept after injection of BHPN in a clean room for 24 months. The incidence of primary lung tumors in rats exposed to 0.05 ppm O3, 0.05 ppm O3 + 0.4 ppm NO2 and 0.4 ppm NO2 + 1 mg/m3 of H2SO4-aerosol with BHPN treatment was 8.3% (3 out of 36 rats), 13.9% (5 out of 36 rats) and 8.3% (3 out of 36 rats), respectively. The tumors were adenomas and adenocarcinomas. The incidence of adenomas was 2.8% (1 out of 36 rats) in the O3 alone group, 11% (4 out of 36 rats) in O3 + NO2 group and 5.6% (2 out of 36 rats) in NO2 + H2SO4 group. The incidence of adenocarcinomas was 5.6% (2 out of 36 rats) in the O3 group, 2.8% (1 out of 36 rats) in O3 + NO2 group and 2.8% (1 out of 36 rats) in NO2 + H2SO4 group. No lung tumors were found in the rats exposed to clean air with BHPN treatment and in animals not given BHPN but exposed to each air pollutant. The difference in tumor incidence between the clean air group with BHPN and the O3 + NO2 group with BHPN was statistically significant. The results show that exposure to O3 alone enhances tumor development and that the combined exposure to O3 or H2SO4 with NO2 produces an additional increase in incidence of lung tumor, respectively. The incidence of slight-moderate to marked alveolar cell hyperplasia in the groups exposed to each air pollutant with BHPN treatment was higher than that in the groups exposed to clean air with BHPN. Exposure to each air pollutant had no effect on the development of bronchiolar mucosal hyperplasia in lungs of rats treated with BHPN.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical effects of combined gases of nitrogen dioxide and ozone. IV. Changes of lipid peroxidation and antioxidative protective systems in rat lungs upon life span exposure.

Lipid peroxide production, antioxidant contents and activities of antioxidative protective enzymes were examined in lungs of rats exposed to clean air (control group), 0.05 ppm O3, 0.05 ppm O3 + 0.04 ppm NO2 and 0.05 ppm O3 + 0.4 ppm NO2 for 22 months. The results were compared with our previous data in rats exposed to 0.04 ppm NO2, 0.4 ppm NO2 and 4 ppm NO2 for their life span (Sagai et al., Toxicol. Appl. Pharmacol., 73, (1984) 444-456). TBA values used as an index of lipid peroxidation in the lungs were increased maximally at 9 months, but were decreased below control values in animals exposed for 18 and 22 months. Nonprotein sulfhydryl (NPSH) contents were increased maximally at 9 months, and after 18 and 22 months were decreased significantly below control values. Vitamin E (VE) contents showed a similar trend. On the other hand, enzyme activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione peroxidase measured by using cumene hydroperoxide (cum.OOH) substrate (GPx-cum.OOH), glutathione peroxidase measured by using H2O2 as a substrate (GPx-H2O2), glutathione S-transferase (GSH-Tase) and superoxide dismutase (SOD) did not show any significant changes during this experiment. The results show that lipid peroxidation in lungs was increased synergistically by a combination of NO2 and O3 at ambient levels, and that the time of maximum lipid peroxide production was shorter than with NO2 alone. The protective ability against lipid peroxides was higher with increased lipid peroxide levels, but the inducibility was not maintained through a life span exposure to the combined gases. Additionally, two small adenomas were observed in 2 out of 18 rats in the 0.05 ppm O3 + 0.04 ppm NO2 group and a large adenoma was observed in 1 out of 18 animals in the 0.05 ppm + 0.4 ppm NO2 group exposed for 22 months.

Experimental studies on tumor promotion by nitrogen dioxide.

The effects of nitrogen dioxide (NO2) on promotion of lung tumorigenesis induced by N-bis(2-hydroxypropyl) nitrosamine (BHPN) were investigated in male Wistar rats. In a preliminary study, the highest non-effective dose of BHPN was found to be 0.5 g per kg body weight. Rats were given a single intraperitoneal injection of BHPN at a dose of 0.5 g per kg body weight or saline at 6 weeks of age, and then exposed to clean air, 0.04 ppm, 0.4 ppm or 4 ppm of NO2 for 17 months, respectively. The incidence of pulmonary tumors in rats exposed to BHPN plus 4 ppm of NO2 was 12.5%; the tumors were adenomas and adenocarcinomas. Adenomas were found in 4 out of 40 rats (10%) and adenocarcinomas were found in 1 out of 40 rats (2.5%). The tumor incidence in the lungs of rats kept in BHPN plus clean air and BHPN plus 0.04 ppm of NO2 was 2.5% (1/40). In both groups adenomas were found. There was no significant difference in tumor incidence between animals exposed to BHPN plus clean air and to BHPN plus 4 ppm of NO2. No lung tumors were found in the group of BHPN plus 0.4 ppm NO2 and in animals exposed to NO2 without BHPN treatment. A high incidence of alveolar cell hyperplasia was observed in the lungs of rats injected with BHPN, and the effect of NO2 on development of alveolar cell hyperplasia was slight. On the other hand, marked bronchiolar mucosal hyperplasia was found in 17 out of 40 rats (42.5%) in the group of BHPN plus 4 ppm of NO2, and in 1 out of 40 rats (2.5%) in each of the group exposed to clean air, 0.04 ppm or 0.4 ppm of NO2 with BHPN treatment, respectively. The hyperplasia in lungs of rats exposed to 4 ppm of NO2 without BHPN treatment was slighter than that in lung of rat exposed to 4 ppm of NO2 with BHPN treatment. On the other hand, tumor incidence in the nasal cavity of rats in each of group exposed to clean air and NO2 with BHPN treatment was 97-100%. Incidence of tumors in other organs in the groups exposed to clean air and NO2 with and without BHPN treatment was very low, and NO2 had no effect on tumor development in the nasal cavity and other organs whether animals were treated with BHPN or not.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Biochemical effects on combined gases of nitrogen dioxide and ozone. I. Species differences of lipid peroxides and phospholipids in lungs.

In the present study, changes of lipid peroxides, phospholipids and antioxidant levels in lungs of 4 animal species exposed to the combined gases of NO2 and O3 were compared. Male mice, hamsters, rats and guinea pigs were used. Lipid peroxides were increased significantly in the lungs of mice and guinea pigs exposed to the combined gases, but not in hamsters and rats. Changes of alpha-tocopherol (VE) contents were slight. On the other hand, non-protein sulfhydryl (NPSH) contents were increased strikingly, especially in hamsters, but were not increased in guinea pigs. Phosphatidylcholine (PC) contents were increased and phosphatidylethanolamine (PE) contents were decreased by the exposure to the combined gases, with the order guinea pig greater than mouse greater than rat. In hamsters no changes were seen. The changes of fatty acid composition in guinea pigs and mice were marked, the increases of palmitate and palmitolate and the decreases of polyunsaturated fatty acid were especially characteristic. These changes in phospholipid class and fatty acid composition may be a "a kind of adaptation phenomenon" to avoid further lipid peroxidation. On the other hand, the changes in hamsters and rats were small. The results show the existence of species differences in lipid peroxide formation by exposure to the combined gases of NO2 and O3. They were found to be related to the contents of antioxidants and the compositions of phospholipids and their fatty acids.

Renal damage induced by cadmium-metallothionein: effects on biochemical indicators.

Time dependent changes in urinary biochemical indicators for renal tubular injury and dysfunction were determined in female Wistar rats after an intraperitoneal injection of cadmium-metallothionein (Cd-MT) (50, 150 or 300 micrograms Cd/kg body wt) to further characterize the tubular damage caused by Cd. The Cd-MT injection caused dose-dependent increases in urinary activities of the enzymes (alkaline phosphatase; gamma-glutamyl transpeptidase; lactate dehydrogenase, LDH; N-acetyl-beta-D-glucosaminidase) on day 1, which appeared to reflect the tubular injury. The rate of increase in LDH, a cytosolic enzyme, was the largest among those of the enzymes. This result coincided with the data reported for repeated administration of ionic Cd to rats, suggesting that the feature of tubular injury caused by an injection of Cd-MT is similar to that by chronic exposure to ionic Cd. Changes in urinary glucose and total protein, indicators of tubular dysfunction, and metals (Cd, zinc and copper) were accompanied with those in urinary enzymes. Hydrocarbons in breath of rats injected with Cd-MT at a dose of 300 micrograms Cd/kg body weight were also determined as an indicator of in vivo lipid peroxidation. The levels of ethane and propane were significantly increased at 12 h after injection, which suggests that lipid peroxidation is partly involved in the tubular damage reflected by the increases in urinary enzymes.

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