Biochemical assessment of acute nitrogen dioxide toxicity in rat lung

The early primary biochemical response of lung to NO2 was studied separately from the later secondary responses of inflammation and proliferation by measuring several biochemical parameters in lungs of rats immediately following a 4-hr exposure to nitrogen dioxide (NO2) at concentrations of 10, 20, 30, and 40 ppm. Cell-free lavage fluid contained elevated amounts of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (GDH), acid phosphatase (AP), and aryl sulfatase (AS) after 30 or 40 ppm NO2. Total protein and sialic acid were increased in cell-free lavage after 20, 30, or 40 ppm NO2. The amounts of protein, sialic acid, and acid phosphatase recovered by airway lavage were equal to the amounts found in 0.7 ml of plasma, consistent with transudation of this volume of plasma into airways as a source of these parameters. The plasma activity of the other parameters measured was too low to account for their increase in lavage fluid by plasma leakage into airways. Decrease in the number and enzyme content of lavagable cells indicated damage to free cells in the airways. The amount of the decrease in enzyme content of the lavagable cell fraction was similar to the increase in the cell-free lavage for all of the measured enzymes except acid phosphatase, suggesting the release of these enzymes into airways as a result of damage to free cells. However, the LDH isoenzyme profile in cell-free lavage after exposure is inconsistent with free cells as the source of this enzyme. No changes were observed in the whole-lung homogenate content of protein, DNA, lipid, LDH, MDH, IDH, GDH, AP, AS, glutathione reductase, NADPH cytochrome c, or succinate cytochrome c reductase immediately after NO2 exposure. This study indicates that initial acute damage to lung by NO2 results in translocation of enzymes, proteins, and sialic acid into airways. Plasma is a likely source of translocated protein, sialic acid, and acid phosphatase. The sources of the other enzyme activities remain to be identified, with lung parenchyma and free cells as likely sources.     

Early damage indicators in the lungs. IV. Biochemical and cytologic response of the lung to lavage with metal salts

A rapid screening test for estimating the acute toxicity level of substances in the lung has been developed and evaluated using metal salts as the toxic agents. In the test, animals were exposed in vivo to the metal salts by bronchopulmonary lavage, and toxicity was determined by the enzymatic and cytologic response observed in the airways 1 day after exposure. The airway response was determined by analysis of bronchopulmonary lavage fluid for lactate dehydrogenase, acid and alkaline phosphatase, and β-glucuronidase activity as well as for total sialic acid and protein content and total and differential cell counts. These values showed a dose-dependent response in the lavage fluid. The enzymatic response of the lung tissue itself was not as marked as that seen in the lavage fluid. Histopathological evaluations of lung tissue were made in order to correlate morphological change with the airway response. The test allowed a rapid determination of the lung dose of metal salt which caused acute toxicity. Based on the biochemical response in the airway, the relative toxicity of the heavy-metal-containing compounds tested was: CdCl₂>SeO₂, NH₄VO₃, NiCl₂>CrCl₃.     

The effect of lung alpha-tocopherol content on the acute toxicity of nitrogen dioxide

The effect of lung vitamin E content on early direct damage to lung by NO2 was studied by exposing three groups of rats differing in lung vitamin E content to 0, 10, 20, 30, and 40 ppm NO2 for 4 hr. Lung vitamin E contents of 3.24, 17.4, and 87.7 micrograms/lung were obtained by maintaining animals on semipurified diets containing 0, 10, or 1000 mg/kg of d-alpha-tocopherol acetate. Animals were sacrificed immediately after the 4-hr exposure and lung damage was assessed by assaying the lung lavage content of protein, sialic acid, lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (GDH), acid phosphatase (AP), and aryl sulfatase (AS), all of which increase in lavage fluid in a concentration-dependent manner over the range of NO2 concentrations used. Increases in lavagable protein, sialic acid, AP, and AS were not affected by the different vitamin E contents, while the increases in LDH, MDH, and GDH were significantly attenuated in the 1000-mg/kg diet group relative to the 0- and 10-mg/kg diet groups. Lipid peroxidation was not detectable in NO2-exposed lungs by either conjugated diene measurement or thiobarbituric-acid-reactive materials, with the exception of a slight increase in thiobarbituric-acid-reactive material in free cells. These results suggest two mechanisms of NO2 damage to lung. The attenuation of the appearance of some lavage parameters by high vitamin E is consistent with lipid peroxidation as a necessary event in the damage responsible for their appearance, although the lack of change in indicators of lipid peroxidation in the whole lung suggests that peroxidation occurs to only a very limited extent. The lavage parameters which are unaffected by lung vitamin E content apparently appear in airways as a result of events not involving lipid peroxidation.     

Effect of pneumotoxicants on lactate dehydrogenase activity in airways of rats

To assess the usefulness of monitoring changes in lactate dehydrogenase (LDH) activity in the cell-free fraction of bronchopulmonary lavage fluid as an indicator of pulmonary injury, several toxicants were administered to rats using treatment regimens which have been shown previously to result in histopathologically demonstrable pneumotoxicity. For all of the toxicants employed except exposure to 1 atm O₂, treatment did not affect recovery of lavage fluid from the lung. Lavage fluid LDH activity was elevated following treatment with paraquat, bromobenzene, and oxygen. Administration of monocrotaline using a treatment regimen which results predominantly in lung injury also produced elevated LDH activity in lavage fluid; however, treatment with this same compound using a regimen which results predominantly in hepatic injury failed to elevate lavage fluid LDH activity but resulted in a more pronounced increase in LDH activity in plasma. Similarly, the hepatotoxicant, carbon tetrachloride, produced a marked increase in plasma LDH activity without affecting that of the lavage fluid. The sensitivity of this method in detecting increased LDH activity was minimally dependent upon the volume of fluid used for lavage or on the number of successive lavages used for analysis. It appears to be useful as a rapid screening test for detecting lung injury from chemicals which are pneumotoxic following inhalation or systemic administration.     

Free lung cell phagocytosis and lysosomal enzyme activity after inhalation of lipopolysaccharide in guinea-pigs

Free lung cells were studied in guinea-pigs different times after an inhalation exposure to bacterial endotoxin yielding a dose of about 1 microgram/animal. After exposure the number of alveolar macrophages (AM) and neutrophils was increased. The phagocytosis capacity of the lung was increased, mainly due to the influx of neutrophils. The activity of N-acetyl-beta-D-glucosaminidase from exposed animals was decreased in AM cell cultures and increased in lavage fluid 2 hours and later after exposure. Cathepsin D activity was also decreased in AM cell cultures but did not change in lavage fluid. Lactate dehydrogenase was increased in lung lavage fluid. The results show that LPS inhalation activates AM and that increased amounts of lysosomal enzymes are present in the airways.     

Inhibition of rat respiratory-tract cytochrome P-450 activity after acute low-level m-xylene inhalation: role in 1-nitronaphthalene toxicity

The xylenes are commonly used industrial solvents that have been shown to inhibit cytochrome P-450 (CYP450) activities in an organ- and isozyme-specific pattern. This study examined the dose-response and durational effects of m-xylene inhalation on cytochrome P-450 activities in the respiratory tract and liver as well as the effects of these CYP450 alterations on 1-nitronaphthalene (1-NN)-induced respiratory or hepatic toxicity. After m-xylene inhalation exposure there was a dose-related inhibition of all nasal mucosa CYPs examined. At 300 ppm, inhibition was sustained up to 2 days after exposure, but on day 5 all CYP activities were increased. There was also dose-related inhibition of lung CYPs 2B1, 2E1, and 4B1. The activities of these CYPs returned to those of control by day 2 but lung CYP 2B1 was increased 5 days following m-xylene exposure. Hepatic CYP 2E1 activity was increased immediately following m-xylene exposure (300 ppm). CYP 2B1 and CYP 1A2 activities were increased through day 2, all activities returning to control values 5 days postexposure. 1-NN treatment caused severe respiratory toxicity that was prevented by prior m-xylene exposure. Lactate dehydrogenase (LDH) and protein were increased in nasal lavage fluid (NLF) but gamma-glutamyl transferase (GGT) was unchanged. m-Xylene coexposure prevented or ameliorated the increases in LDH and protein but increased GGT. 1-NN-induced increases in bronchoalveolar lavage fluid (BALF) LDH and GGT were attenuated by m-xylene. 1-NN caused pronounced histopathological changes in both respiratory and olfactory regions of the nasal mucosa. Lesions in both regions were characterized by acute epithelial necrosis and exfoliation and suppurative exudate in the airways. These changes were prevented by m-xylene coexposure. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were not changed in animals exposed to 1-NN but were increased by m-xylene coexposure. Low-level m-xylene exposure organ-selectively altered CYP450 isozyme activities and subsequent 1-NN toxicity.     

Lactate dehydrogenase isoenzymes in hamster lung lavage fluid after lung injury

Lactate dehydrogenase (LD) levels and isoenzyme patterns were determined in the cell-free supernatant fractions of lung lavage fluid from hamsters exposed to alpha-quartz, iron oxide, Triton X-100, 100% O₂, or 200 ppm SO₂. The isoenzyme patterns were compared to those derived from hamster lung homogenates, serum, polymorphonuclear neutrophils (PMNs), pulmonary macrophages, and red blood cells. The isoenzyme patterns from alpha-quartz- and iron oxide-exposed animals resembled each other and were similar to that of PMNs. In contrast, the pattern seen after Triton X-100 exposure was similar to those of whole lung homogenates and of red blood cells. A 96-hr exposure to 100% O₂ yielded an LD isoenzyme pattern in lung lavage fluid similar to that of serum. Exposure to SO₂ did not alter LD levels, showing that upper airways damage is not reflected by changes in LD in lung lavage fluid. We conclude that LD isoenzyme patterns of lung lavage fluid can be used to differentiate among types of pulmonary injury and may help identify the sites of injury.     

The effect of dietary restriction and altered sodium intake on the cardiopulmonary toxicity of monocrotaline pyrrole

Monocrotaline pyrrole (MCTP) is a reactive metabolite of the plant toxin monocrotaline (MCT), which produces pulmonary vascular injury and right ventricular hypertrophy in rats. In this study, the influence of diet restriction on the cardiopulmonary toxicity of MCTP was examined. In rats fed ad libitum, MCTP treatment resulted in increased lung weight, in elevated lactate dehydrogenase (LDH) activity and protein concentration in cell-free bronchopulmonary lavage fluid, and in right ventricular enlargement. Restriction of feed intake to 40% of normal attenuated the increases in lung weight and lavage protein concentration in MCTP-treated rats and abolished the right ventricular enlargement but did not affect the increased lavage LDH activity. In a study of the effect of diet restriction on the survival of MCTP-treated rats, the percentage of diet-restricted animals surviving was significantly higher than that of surviving animals which ate ad libitum through Day 28, but thereafter there was no significant difference between the two groups. Alterations in dietary sodium intake alone did not affect MCTP-induced toxicity. These results indicate that diet restriction partially protects against the cardiopulmonary toxicity due to MCTP, and that this protective effect cannot be explained by changes in salt intake.     

EFFECT OF INHALATION OF LOW DOSES OF CROCIDOLITE AND FIBROUS GYPSUM ON THE GLUTATHIONE CONCENTRATION AND-GLUTAMYL TRANSPEPTIDASE ACTIVITY IN MACROPHAGES AND BRONCHOALVEOLAR LAVAGE FLUID

Inhalation of asbestos can result in pulmonary fibrosis and in some cases the development of tumors, often many years after exposure. There is, however, an acute response by the lung, particularly the lower respiratory tract, which is part of the defensive mechanism for clearing the fibers. This is a process dominated by alveolar macrophages, which phagocytose the shorter fibers and release inflammatory products in attempting to deal with the longer ones. The result of this can be the production of active oxygen species that may lead to oxidative damage. To defend against such injury, the lung airways and parenchyma contain a number of antioxidants for their protection, of which the dominant one is glutathione (GSH). An alteration in levels of GSH as a result of toxic action could leave the lung vulnerable to damage. GSH is present at high concentration in Clara cells, type II pneumocytes, and alveolar macrophages, and there is also a high extracellular concentration of the compound. (-Glutamyl transpeptidase (-GT), the enzyme responsible for the breakdown of GSH prior to uptake, is found in the membranes of Clara and type II cells of the rat lung, although only negligible amounts are found in macrophages. This study investigated the effect of inhaled crocidolite asbestos on GSH concentration (in the form of nonprotein sulfydryl, NPSH) and-GT activity in macrophages and bronchoalveolar lavage (thought to represent the extracellular milieu). Calcined anhydrous gypsum was used as a putative control. We have previously studied similar endpoints using high exposure levels. This study, however, used dose levels closer to that seen in heavily industrially exposed individuals. We found that NPSH levels were significantly increased in both macrophages and acellular bronchoalveolar lavage fluid after 4 wk of exposure to crocidolite. The levels were still high after a 4-wk recovery period.-GT only altered significantly in lavage, but this occurred after exposure to both calcium sulfate and crocidolite. This response is contrary to that obtained in the previous investigation using high exposure levels. Both studies test the hypothesis that GSH would be mobilized by these cell types under a toxic insult. We conclude that exposure to lower levels of crocidolite causes a significant increase in intra- and extracellular NPSH. It is not possible to say from the present data whether the increase in extracellular NPSH has arisen from epithelial cell secretion or was caused by macrophage phagocytosis. However, the presence of such high amounts of antioxidant in the airways may prove to be pertinent to the mechanism of fiber toxicity.     

Toxicity of cadmium oxide instilled into the rat lung. II. Inflammatory responses in broncho-alveolar lavage fluid

Biochemical and cytological responses in the broncho-alveolar lavage fluid were investigated after instillation of cadmium oxide (CdO) or cadmium chloride (CdCl2) into the rat lung. Although biochemical responses of the lung to CdO were similar to the CdCl2-exposed lung, cytological response was more sensitive to CdO than CdCl2. Increases of lactate dehydrogenase, protein content and number of cells in the lavage fluid were proportional to the dose over the range of 0.5-10 micrograms Cd/rat. beta-Glucuronidase activity in the fluid increased with dose at low doses of Cd, but the activity did not continue to increase above 2 micrograms Cd/rat. A dose-response profile of phosphorus content in the lavage fluid, which might indicate amount of surfactant produced by Type II cells was similar to that observed for beta-glucuronidase in CdO-treated rats. Thus, tolerable level of instilled CdO for the rat lung was about 2 micrograms Cd/rat.     

Bronchoalveolar lavage in rats and mice following beryllium sulfate inhalation

The effects of lung injury in rats and mice exposed to an aerosol of beryllium sulfate for 1 hr through nose-only inhalation were evaluated by the method of bronchoalveolar lavage. The lavage fluid of rats exposed to an aerosol of either 3.3 or 7.0 micrograms Be/liter over a 21-day period following exposure indicated lactate dehydrogenase (LDH) and alkaline phosphatase (Alk Pase) activities to be the most sensitive indicators of lung damage. LDH activity peaked at 8 days postexposure while Alk Pase activity was maximum at Day 5. Both values were 30 times greater than comparable controls at these time points. Acid phosphatase activity and albumin levels also increased over the 21-day period, but not to the same extent. The lung lavage of mice exposed to 7.2 micrograms Be/liter showed LDH activity as the most sensitive indicator of lung damage with a maximum response three times greater than that of controls at Day 5.     

Ovalbumin-Induced Airway Inflammation and Fibrosis in Mice Also Exposed to Ozone

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ozone on airway inflammation and remodeling. Sensitized BALB/c mice were exposed to ovalbumin aerosol for 4 wk before and after 2 wk of exposure to either 0.2 ppm or 0.5 ppm ozone. Other groups of mice were exposed to ovalbumin aerosol for 6 wk with continuous concurrent exposure to ozone during wk 1–6, or during intermittent concurrent exposure to ozone. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells and by histological evaluation of stained lung sections. Alterations in lung structure (airway fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after exposure to 2 wk of 0.2 or 0.5 ppm ozone. Mice exposed to ovalbumin for 6 wk with concurrent exposure to either 0.2 ppm or 0.5 ppm ozone during wk 3–6 had a significant decrease in the total number of cells recovered by lavage. Values as low as 7% of the cell number found in mice exposed to ovalbumin aerosol alone were observed in the mice exposed to ovalbumin plus 0.2 ppm ozone during wk 3–6. There were significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and ozone under all of the protocols studied. When ozone was given for 2 wk prior to ovalbumin exposure (Experiment 1), there were a high percentage of macrophages and low percentages of lymphocytes and eosinophils in the lung lavage. When ozone was given for 2 wk after ovalbumin exposure (Experiment 2), there were a moderate percentage of macrophages, a low percentage of eosinophils, and a high percentage of lymphocytes in the lung lavage. When ozone and ovalbumin were given simultaneously (Experiments 3 and 4), there were a high percentage of macrophages in the lavage with 0.2 ppm ozone and a high percentage of eosinophils. Ozone appears to antagonize the specific inflammatory effects of ovalbumin exposure, especially when given before or during exposure to ovalbumin. Airway remodeling was examined by two different quantitative methods. None of the groups exposed concurrently to ovalbumin and ozone had a significant increase in airway collagen content as compared to the matched groups of mice exposed to ovalbumin alone. The findings were consistent with an additive response of mice to simultaneous exposure to ovalbumin and ozone. Ozone exposure alone for 6 wk did not affect the number of goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 6 wk had about 25% goblet cells in their conducting airways. Concurrent exposure to ovalbumin and 0.2 ppm ozone caused significant increases in goblet cells (to 43% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to ozone, the lung inflammatory response may be modified, but that this altered response is dependent on the sequence of exposure and the concentration of ozone to which they are exposed. At the concentrations of ozone tested, we did not see changes in airway fibrosis. However, goblet-cell hyperplasia appeared to be increased in mice exposed concurrently to ovalbumin and 0.2 ppm ozone.     

Residual oil fly ash inhalation in guinea pigs: influence of absorbate and glutathione depletion.

Inhaled urban particulate matter (PM) often contains metals that appear to contribute to its toxicity. These particles first make contact with a thin layer of epithelial lining fluid in the respiratory tract. Antioxidants present in this fluid and in cells might be important susceptibility factors in PM toxicity. We investigated the role of ascorbic acid (C) and glutathione (GSH) as determinants of susceptibility to inhaled residual oil fly ash (ROFA) in guinea pigs (male, Hartley). Guinea pigs were divided into four groups, +C+GSH, +C-GSH, -C+GSH, and -C-GSH, and exposed to clean air or ROFA (< 2.5 micron diameter, 19--25 mg/m(3) nose-only for 2.0 h). C and/or GSH were lowered by either feeding C-depleted diet (1 microg C/kg diet, 2 weeks) and/or by ip injection of a mixture of buthionine-S,R-sulfoximine (2.7 mmol/kg body weight) and diethylmaleate (1.2 mmol/kg, 2 h prior). Nasal lavage (NL) and bronchoalveolar lavage (BAL) fluid and cells were examined at 0 h and 24 h postexposure to ROFA. The C-deficient diet lowered C concentrations in BAL fluid and cells and in NL fluid by 90%, and the GSH-depletion regimen lowered both GSH and C in the BAL fluid and cells by 50%. ROFA deposition was calculated at time 0 from lung Ni levels to be 46 microg/g wet lung. In unexposed animals, the combined deficiency of C and GSH modified the cellular composition of cells recovered in lavage fluid, i.e., the increased number of eosinophils and macrophages in BAL fluid. ROFA inhalation increased lung injury in the -C-GSH group only (evidenced by increased BAL protein, LDH and neutrophils, and decreased BAL macrophages). ROFA exposure decreased C in BAL and NL at 0 h, and increased BAL C and GSH (2- to 4-fold above normal) at 24 h in nondepleted guinea pigs, but had no effect on C and GSH in depleted guinea pigs. Combined deficiency of C and GSH resulted in the highest macrophage and eosinophil counts of any group. GSH depletion was associated with increased BAL protein and LDH, increased numbers of BAL macrophages and eosinophils, and decreased rectal body temperatures. We conclude that combined deficiency of C and GSH increased susceptibility to inhaled ROFA; caused unusual BAL cellular changes; resulted in lower antioxidant concentrations in BAL than were observed with single deficiencies. Antioxidant deficiency may explain increased susceptibility to PM in elderly or diseased populations and may have important implications for extrapolating animal toxicity data to humans.     

Acute inhalation toxicity of 2-chloro-1,3-butadiene (chloroprene): Effects on liver and lung

2-Chloro-1,3-butadiene (chloroprene, CBD), a major industrial chemical used in synthetic chlorinated rubber production, is acutely hepatotoxic. Fasted rats were exposed to concentrations of 100, 150, 225, and 300 ppm CBD for 4 hr and killed at 24 hr. Liver injury as measured by increased serum sorbitol dehydrogenase (SDH) activity was apparent in animals exposed to 225 and 300 ppm CBD. Non-protein sulfhydryl (NPSH) concentrations in liver were increased 24 hr after all exposures. Lung NPSH concentrations were decreased significantly after 100- and 300-ppm exposure. Serum lactate dehydrogenase (LDH) activity was increased after exposure to 300 ppm. When this same enzyme was measured in lung lavage fluid from animals killed 24 hr after exposure, no elevation was detected. Thus, acute lung injury did not manifest itself with a release of LDH into the airway at this time. Alternatively, airway injury may have resolved by 24 hr. Aroclor 1254 (PCB) pretreatment prevented liver injury (SDH elevation, liver enlargement, and NPSH rebound) following exposure to 100 and 300 ppm CBD. Lung NPSH was not decreased by CBD exposure of PCB-pretreated, fasted rats. The pattern of CBD toxicity is comparable to 1,1-dichloroethylene toxicity.     

Structural defects play a major role in the acute lung toxicity of multiwall carbon nanotubes: toxicological aspects

Experimental studies indicate that carbon nanotubes (CNTs) have the potential to induce adverse pulmonary effects, including alveolitis, fibrosis, and genotoxicity in epithelial cells. Here, we explored the physicochemical determinants of these toxic responses with progressively and selectively modified CNTs: ground multiwall CNTs modified by heating at 600 degrees C (loss of oxygenated carbon functionalities and reduction of oxidized metals) or at 2400 degrees C (annealing of structural defects and elimination of metals) and by grinding the material that had been heated at 2400 degrees C before (introduction of structural defects in a metal-deprived framework). The CNTs were administered intratracheally (2 mg/rat) to Wistar rats to evaluate the short-term response (3 days) in bronchoalveolar lavage fluid (LDH, proteins, cellular infiltration, IL-1beta, and TNF-alpha). The long-term (60 days) lung response was assessed biochemically by measuring the lung hydroxyproline content and histologically. In vitro experiments were also performed on rat lung epithelial cells to assess the genotoxic potential of the modified CNTs with the cytokinesis block micronucleus assay. The results show that the acute pulmonary toxicity and the genotoxicity of CNT were reduced upon heating but restored upon grinding, indicating that the intrinsic toxicity of CNT is mainly mediated by the presence of defective sites in their carbon framework.     

Interstitial and free lung cells in acute inflammation in the guinea-pig

Different cell types were studied in bronchoalveolar lavage fluid (BAL) and solid lung tissue of guinea-pigs. Whereas alveolar macrophages (AM) and eosinophils predominated in BAL, the proportion of AM and lymphocytes was highest in the lung tissue. After an inhalation exposure to LPS, the number of neutrophils increased rapidly in the lung tissue reaching a maximum after 4 hours, and more slowly in the airways reaching a maximum after 24 hours. This suggests that other mechanisms than secretion of chemotactic factors from AM, shown to be active up to 4 hours after exposure, are responsible for the later phase of the neutrophil invasion into the airways. Passive migration or other mediators may be involved.     

Ovalbumin-induced airway inflammation and fibrosis in mice also exposed to ozone

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ozone on airway inflammation and remodeling. Sensitized BALB/c mice were exposed to ovalbumin aerosol for 4 wk before and after 2 wk of exposure to either 0.2 ppm or 0.5 ppm ozone. Other groups of mice were exposed to ovalbumin aerosol for 6 wk with continuous concurrent exposure to ozone during wk 1-6, or during intermittent concurrent exposure to ozone. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells and by histological evaluation of stained lung sections. Alterations in lung structure (airway fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after exposure to 2 wk of 0.2 or 0.5 ppm ozone. Mice exposed to ovalbumin for 6 wk with concurrent exposure to either 0.2 ppm or 0.5 ppm ozone during wk 3-6 had a significant decrease in the total number of cells recovered by lavage. Values as low as 7% of the cell number found in mice exposed to ovalbumin aerosol alone were observed in the mice exposed to ovalbumin plus 0.2 ppm ozone during wk 3-6. There were significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and ozone under all of the protocols studied. When ozone was given for 2 wk prior to ovalbumin exposure (Experiment 1), there were a high percentage of macrophages and low percentages of lymphocytes and eosinophils in the lung lavage. When ozone was given for 2 wk after ovalbumin exposure (Experiment 2), there were a moderate percentage of macrophages, a low percentage of eosinophils, and a high percentage of lymphocytes in the lung lavage. When ozone and ovalbumin were given simultaneously (Experiments 3 and 4), there were a high percentage of macrophages in the lavage with 0.2 ppm ozone and a high percentage of eosinophils. Ozone appears to antagonize the specific inflammatory effects of ovalbumin exposure, especially when given before or during exposure to ovalbumin. Airway remodeling was examined by two different quantitative methods. None of the groups exposed concurrently to ovalbumin and ozone had a significant increase in airway collagen content as compared to the matched groups of mice exposed to ovalbumin alone. The findings were consistent with an additive response of mice to simultaneous exposure to ovalbumin and ozone. Ozone exposure alone for 6 wk did not affect the number of goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 6 wk had about 25% goblet cells in their conducting airways. Concurrent exposure to ovalbumin and 0.2 ppm ozone caused significant increases in goblet cells (to 43% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to ozone, the lung inflammatory response may be modified, but that this altered response is dependent on the sequence of exposure and the concentration of ozone to which they are exposed. At the concentrations of ozone tested, we did not see changes in airway fibrosis. However, goblet-cell hyperplasia appeared to be increased in mice exposed concurrently to ovalbumin and 0.2 ppm ozone.     

Lung antioxidant and cytokine responses to coarse and fine particulate matter from the great California wildfires of 2008

The authors have previously demonstrated that wildfire-derived coarse or fine particulate matter (PM) intratracheally instilled into lungs of mice induce a strong inflammatory response. In the current study, the authors demonstrate that wildfire PM simultaneously cause major increases in oxidative stress in the mouse lungs as measured by decreased antioxidant content of the lung lavage supernatant fluid 6 and 24?h after PM administration. Concentrations of neutrophil chemokines/cytokines and of tumor necrosis factor (TNF)-α were elevated in the lung lavage fluid obtained 6 and 24?h after PM instillation, consistent with the strong neutrophilic inflammatory response observed in the lungs 24?h after PM administration, suggesting a relationship between the proinflammatory activity of the PM and the measured level of antioxidant capacity in the lung lavage fluid. Chemical analysis shows relatively low levels of polycyclic aromatic hydrocarbons compared to published results from typical urban PM. Coarse PM fraction is more active (proinflammatory activity and oxidative stress) on an equal-dose basis than the fine PM despite its lower content of polycyclic aromatic hydrocarbons. There does not seem to be any correlation between the content of any specific polycyclic aromatic hydrocarbon (or of total polycyclic aromatic hydrocarbon content) in the PM fraction and its toxicity. However, the concentrations of the oxidation products of phenanthrene and anthracene, phenanthraquinone and anthraquinone, were several-fold higher in the coarse PM than the fine fraction, suggesting a significant role for atmospheric photochemistry in the formation of secondary pollutants in the wildfire PM and the possibility that such secondary pollutants could be significant sources of toxicity in the wildfire PM.     

Arginases I and II in lungs of ovalbumin-sensitized mice exposed to ovalbumin: sources and consequences

Arginase gene expression in the lung has been linked to asthma both in clinical studies of human patients and in the well-studied mouse model of ovalbumin-induced airway inflammation. Arginase is thought to regulate NO levels in the lung by its ability to divert arginine, the substrate for nitric oxide synthases that produce citrulline and NO, into an alternative metabolic pathway producing ornithine and urea. In the present study arginase I and arginase II concentrations were measured in isolated microdissected airway preparations from sensitized Balb/c mice exposed to ovalbumin aerosol. We found that arginase II was constitutively expressed in the airways of normal mice, whereas arginase I was undetectable in normal airways, while its expression was increased in airways of mice exposed to ovalbumin. The expression of arginase I strongly correlated with the presence of lung inflammation, as quantified by differential cell counts in lung lavage, suggesting that most, or all, of the arginase I in lungs of mice exposed to ovalbumin is present in the inflammatory cells rather than in the airway epithelium. There was also a significant correlation between increased expression of arginase I in the isolated airways and decreased lung compliance. On the other hand, while we found arginase II expression to also be significantly increased in airways from mice exposed to ovalbumin as compared with normal airways, the relative increase was much less than that observed for arginase I, suggesting that there was a smaller contribution of inflammatory cells to the arginase II content of the airways in mice exposed to ovalbumin. There was no apparent correlation between the content of arginase in isolated airways and exhaled NO concentration in the expired air from mice exposed to ovalbumin. However, there was a correlation between exhaled NO concentration from mice exposed to ovalbumin and the lymphocyte content of the lung lavage. The concentration of arginine found in isolated airways from Balb/c mice exposed for 2 weeks to ovalbumin was about half of the value found in isolated microdissected airways from normal mice. Treatment of mice systemically with an arginase inhibitor significantly increased the amount of NO produced, as measured as the amount of nitrite+nitrate (NOx) in lung lavage supernatant prepared from mice exposed to ovalbumin. Our results are consistent with the hypothesis that the response of the lung to ovalbumin challenge includes an adaptive response in the large airways regulating the concentration of arginine within cells of the airway epithelium and subepithelial layer, by shunting of arginine into the metabolic pathway for increased synthesis of NO.     

Inhibition of Rat Respiratory-Tract Cytochrome P-450 Activity After Acute Low-Level m-Xylene Inhalation: Role in 1-Nitronaphthalene Toxicity

The xylenes are commonly used industrial solvents that have been shown to inhibit cytochrome P-450 (CYP450) activities in an organ- and isozyme-specific pattern. This study examined the dose-response and durational effects of m-xylene inhalation on cytochrome P-450 activities in the respiratory tract and liver as well as the effects of these CYP450 alterations on 1-nitronaphthalene (1-NN)-induced respiratory or hepatic toxicity. After m-xylene inhalation exposure there was a dose-related inhibition of all nasal mucosa CYPs examined. At 300 ppm, inhibition was sustained up to 2 days after exposure, but on day 5 all CYP activities were increased. There was also dose-related inhibition of lung CYPs 2B1, 2E1, and 4B1. The activities of these CYPs returned to those of control by day 2 but lung CYP 2B1 was increased 5 days following m-xylene exposure. Hepatic CYP 2E1 activity was increased immediately following m-xylene exposure (300 ppm). CYP 2B1 and CYP 1A2 activities were increased through day 2, all activities returning to control values 5 days postexposure. 1-NN treatment caused severe respiratory toxicity that was prevented by prior m-xylene exposure. Lactate dehydrogenase (LDH) and protein were increased in nasal lavage fluid (NLF) but gamma-glutamyl transferase (GGT) was unchanged. m-Xylene coexposure prevented or ameliorated the increases in LDH and protein but increased GGT. 1-NN-induced increases in bronchoalveolar lavage fluid (BALF) LDH and GGT were attenuated by m-xylene. 1-NN caused pronounced histopathological changes in both respiratory and olfactory regions of the nasal mucosa. Lesions in both regions were characterized by acute epithelial necrosis and exfoliation and suppurative exudate in the airways. These changes were prevented by m-xylene coexposure. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were not changed in animals exposed to 1-NN but were increased by m-xylene coexposure. Low-level m-xylene exposure organ-selectively altered CYP450 isozyme activities and subsequent 1-NN toxicity.