Iron disequilibrium in the rat lung after instilled blood.

STUDY OBJECTIVES: The extravasation of erythrocytes into the human lung occurs in a myriad of pulmonary disorders. Metal that is initially included in hemoglobin has been postulated to precipitate a disequilibrium in iron metabolism, to present an oxidative stress, and to contribute to tissue injury in several lung diseases. The objective of this study is to test the hypothesis that the tracheal instillation of blood in an animal model would have significant effects on iron equilibrium and would be associated with an injury to the lower respiratory tract. DESIGN: Rats were intratracheally instilled with either 1.0 mL saline solution (n = 36) or 1.0 mL blood (n = 36). Biochemical end points and histochemistry were obtained at times between 20 min and 14 days after the exposure to saline solution or blood. RESULTS: Total and nonheme iron concentrations in tracheal lavage fluid increased after the instillation of the blood. The percentage of neutrophils in the lavage fluid was elevated 1 day after the instillation of blood and remained at that level for at least 4 days following exposure, while protein concentrations were significantly increased at 1 day and 2 days only. Erythrocytes in the lung tissue were stained for hemoglobin immediately after exposure, but by 4 days after exposure, there was none. Ferritin was elevated between 1 day and 4 days after exposure, but by 7 days after exposure, the expression of this storage protein had returned to baseline values. CONCLUSIONS: We conclude that intratracheal instillation of whole blood in the rat can induce a neutrophilic lung injury that is associated with a disruption of normal iron metabolism. This disruption of the iron equilibrium is made evident by quantifying iron and staining for hemoglobin and ferritin. All indexes of biological effect had corrected by 7 days after exposure.

Ozone adaptation in mice and its association with ascorbic acid in the lung.

We have previously shown that ozone (O(3)) adaptation occurred in rats after daily exposure to an "urban-type" concentration. The adaptation was positively associated with an excess of ascorbic acid (AA) in bronchoalveolar lavage fluid (BALF), suggesting that AA may play a role in the adaptation mechanism. This relationship was not seen at higher and more toxic exposures. The present work exposed mice to low and high levels of O(3) to see if the adaptation-AA relationship is common among rodent species. Male CD-1 mice were studied during repeated 6-h/day exposures to 0.0 or 0.25 ppm O(3) for 10 days and 10 days of recovery in air (experiment 1) and to 0.0, 0.5, or 1.0 ppm O(3) for 5 days (experiment 2). Approximately 20 h after each daily exposure, groups of mice were randomly selected from each concentration type and examined for patterns of response. They were anesthetized (urethane, ip), intubated, and the lungs were lavaged with 37 degrees C saline. BALF was assayed for cells, cell differential, protein, albumin, lactate dehydrogenase, lysozymes, N-acetyl-beta-D-glucosaminidase, gamma-glutamyl transferase, uric acid, glutathione, and AA. Body weight and total lung capacity were also measured. Mice from experiment 1 (10/exposure) were tested for adaptation on day 12 by challenging them with 1.0 ppm O(3) for 6 h and collecting BALF 20 h later. In experiment 2, adaptation was assessed by evaluating the attenuation in response to continued exposure. There was only minimal response to the daily O(3) exposures in experiment 1 except for AA, which was significantly increased in BALF by day 3 and remained elevated well into the recovery period. The O(3)-preexposed mice demonstrated adaptation when compared to their O(3)-naive counterparts. Daily exposure to 1. 0 ppm O(3) in experiment 2 caused weight loss and changes in BALF consistent with toxicity, and neither adaptation nor an excess quantity of AA was seen. The findings in mice were in agreement with those seen in rats and suggest that there may be a common O(3) adaptation mechanism among rodents that involves the regulation of AA in lung lining fluid.

The spontaneously hypertensive rat as a model of human cardiovascular disease: evidence of exacerbated cardiopulmonary injury and oxidative stress from inhaled emission particulate matter.

Cardiovascular disease is considered a probable risk factor of particulate matter (PM)-related mortality and morbidity. It was hypothesized that rats with hereditary systemic hypertension and underlying cardiac disease would be more susceptible than healthy normotensive rats to pulmonary injury from inhaled residual oil fly ash (ROFA) PM. Eight spontaneously hypertensive (SH) and eight normotensive Wistar-Kyoto (WKY) rats (12-13 weeks old) were implanted with radiotelemetry transmitters on Day -10 for measurement of electrocardiographic (ECG) waveforms. These and other nonimplanted rats were exposed to filtered air or ROFA (containing leachable toxic levels of metals) on Day 0 by nose-only inhalation (ROFA, 15 mg/m(3) x 6 h/day x 3 days). ECGs were monitored during both exposure and nonexposure periods. At 0 or 18 h post-ROFA exposure, rats were assessed for airway hyperreactivity, pulmonary and cardiac histological lesions, bronchoalveolar lavage fluid (BALF) markers of lung injury, oxidative stress, and cytokine gene expression. Comparisons were made in two areas: (1) underlying cardiopulmonary complications of control SH rats in comparison to control WKY rats; and (2) ROFA-induced cardiopulmonary injury/inflammation and oxidative burden. With respect to the first area, control air-exposed SH rats had higher lung and left ventricular weights when compared to age-matched WKY rats. SH rats had hyporeactive airways to acetylcholine challenge. Lung histology revealed the presence of activated macrophages, neutrophils, and hemorrhage in control SHrats. Consistently, levels of BALF protein, macrophages, neutrophils, and red blood cells were also higher in SH rats. Thiobarbituric acid-reactive material in the BALF of air-exposed SH rats was significantly higher than that of WKY rats. Lung inflammation and lesions were mirrored in the higher basal levels of pulmonary cytokine mRNA expression. Cardiomyopathy and monocytic cell infiltration were apparent in the left ventricle of SH rats, along with increased cytokine expression. ECG demonstrated a depressed ST segment area in SH rats. With regard to the second area of comparison (ROFA-exposed rats), pulmonary histology indicated a slightly exacerbated pulmonary lesions including inflammatory response to ROFA in SH rats compared to WKY rats and ROFA-induced increases in BALF protein and albumin were significantly higher in SH rats than in WKY rats. In addition, ROFA caused an increase in BALF red blood cells in SH rats, indicating increased hemorrhage in the alveolar parenchyma. The number of alveolar macrophages increased more dramatically in SH rats following ROFA exposure, whereas neutrophils increased similarly in both strains. Despite greater pulmonary injury in SH rats, ROFA-induced increases in BALF GSH, ascorbate, and uric acid were attenuated when compared to WKY rats. ROFA inhalation exposure was associated with similar increases in pulmonary mRNA expression of IL-6, cellular fibronectin, and glucose-6-phosphate dehydrogenase (relative to that of beta-actin) in both rat strains. The expression of MIP-2 was increased in WKY but attenuated in SH rats. Thus, SH rats have underlying cardiac and pulmonary complications. When exposed to ROFA, SH rats exhibited exacerbated pulmonary injury, an attenuated antioxidant response, and acute depression in ST segment area of ECG, which is consistent with a greater susceptibility to adverse health effects of fugitive combustion PM. This study shows that the SH rat is a potentially useful model of genetically determined susceptibility with pulmonary and cardiovascular complications.

Diminished injury in hypotransferrinemic mice after exposure to a metal-rich particle.

Using the hypotransferrinemic (Hp) mouse model, we studied the effect of altered iron homeostasis on the defense of the lung against a catalytically active metal. The homozygotic (hpx/hpx) Hp mice had greatly diminished concentrations of both serum and lavage fluid transferrin relative to wild-type mice and heterozygotes. Fifty micrograms of a particle containing abundant concentrations of metals (a residual oil fly ash) was instilled into wild-type mice and heterozygotic and homozygotic Hp animals. There was an oxidative stress associated with particle exposure as manifested by decreased lavage fluid concentrations of ascorbate. However, rather than an increase in lung injury, diminished transferrin concentrations in homozygotic Hp mice were associated with decreased indexes of damage, including concentrations of relevant cytokines, inflammatory cell influx, lavage fluid protein, and lavage fluid lactate dehydrogenase. Comparable to other organs in the homozygotic Hp mouse, siderosis of the lung was evident, with elevated concentrations of lavage fluid and tissue iron. Consequent to these increased concentrations of iron, proteins to store and transport iron, ferritin, and lactoferrin, respectively, were increased when assayed by immunoprecipitation and immunohistochemistry. We conclude that the lack of transferrin in Hp mice did not predispose the animals to lung injury after exposure to a particle abundant in metals. Rather, these mice demonstrated a diminished injury that was associated with an increase in the metal storage and transport proteins.

Depletion of iron and ascorbate in rodents diminishes lung injury after silica.

Exposures of the lung to iron chelates can be associated with an injury. The catalysis of oxygen-based free radicals is postulated to participate in this injury. Such oxidant generation by mineral oxide particles can be dependent on availability of both iron and a reductant. We tested the study hypothesis that lung injury after silica is associated with the availability of both iron and ascorbate in the host by depleting this metal and reductant in the lungs of rats and guinea pigs, respectively. Rats were fed either a normal diet or a diet deficient of iron. After 30 days, animals were instilled with either saline or 1.0 mg Minusil-5 silica. Relative to saline, silica significantly increased neutrophils and lavage protein. Iron depletion significantly diminished both the cellular influx and injury but only at 1 week after silica exposure. Guinea pigs were provided either a normal diet supplemented with 1,000 ppm vitamin C or a diet deficient in ascorbate. After 14 days, the guinea pigs were instilled with either saline or 1.0 mg silica. Silica exposure significantly increased neutrophils and lavage protein. Ascorbate depletion significantly diminished the influx of inflammatory cells and injury at both 1 day and 1 week after silica exposure. We conclude that host concentrations of both iron and ascorbate can affect lung injury after silica exposure.

Ozone toxicity in the rat. III. Effect of changes in ambient temperature on pulmonary parameters.

Pulmonary toxicity of ozone (O3) was examined in adult male Fischer 344 rats exposed to 0.5 parts/million O3 for either 6 or 23 h/day over 5 days while maintained at an ambient temperature (Ta) of either 10, 22, or 34 degrees C. Toxicity was evaluated by using changes in lung volumes and the concentrations of constituents of bronchoalveolar lavage fluid that signal lung injury and/or inflammation. Results indicated that toxicity increased as Ta decreased. Exposures conducted at 10 degrees C were associated with the greatest decreases in body weight and total lung capacity and the greatest increases in lavageable protein, lysozyme, alkaline phosphatase activity, and percent neutrophils. O3 effects not modified by Ta included increases in residual volume and lavageable potassium, glucose, urea, and ascorbic acid with exposure at 34 degrees C. Most effects were attenuated during the 5 exposure days and/or returned to normal levels after 7 air recovery days, regardless of prior O3 exposure or Ta. It is possible that Ta-induced changes in metabolic rate may have altered ventilation and, therefore, the O3 doses among rats exposed at the three different Ta levels.

Antioxidants in bronchoalveolar lavage fluid cells isolated from ozone--exposed normal and ascorbate-deficient guinea pigs.

Previous studies have indicated that systemic deficiency in one of the critical antioxidants, ascorbate, does not significantly exacerbate ozone-induced lung injury and changes in lung antioxidants following longer-term exposure. Because alveolar cells encounter the highest ozone dose upon exposure and lack direct blood supply, systemic ascorbate deficiency may exacerbate ozone response on antioxidants within these cells. Female Hartley guinea pigs (30 days old) were fed either a regular guinea pig chow or chow that lacked ascorbate. The dietary regimen was started 1 week prior to exposure, continued through ozone exposure (0, 0.2, 0.4, or 0.8 ppm, 23 h/day, 1 week), and during 1 week recovery in clean air following exposure. Immediately after 1 week of exposure or recovery, lungs were lavaged and cells were counted in bronchoalveolar lavage fluid (BALF). Protein, ascorbate, uric acid, total glutathione (GSH), and alpha-tocopherol were analyzed in these cells. Ozone caused an increase in total BALF cells and total cellular protein after 0.4 and 0.8 ppm ozone. The increase was more pronounced in ascorbate-deficient guinea pigs. Protein per million cells, however, was not changed by ozone or diet. In ascorbate-sufficient guinea pigs, ascorbate levels were increased only after 0.2 ppm ozone. However, uric acid (at 0.4 and 0.8 ppm ozone) and GSH (at all concentrations of ozone) levels were increased in both dietary groups. Ascorbate deficiency did not affect basal uric acid or GSH levels in BALF cells. There was a small diet-related depletion in cellular alpha-tocopherol. Ozone exposure also decreased alpha-tocopherol regardless of diet. The above changes except for alpha-tocopherol appeared to be reversed after 1 week of recovery in both dietary groups. In summary, ozone is capable of inducing a mechanism that increases antioxidants such as ascorbate, GSH, and uric acid. GSH and uric acid are not affected by ascorbate deficiency, but alpha-tocopherol is depleted. GSH and uric acid may be critical in ozone-induced adaptation during ascorbate deficiency.

Adaptation to ozone in rats and its association with ascorbic acid in the lung.

Ozone (O3) adaptation is a well-known, but poorly understood phenomenon that has been demonstrated in humans and laboratory animals. This study examined pulmonary function and bronchoalveolar lavage fluid (BALF) parameters in O3-adapted F-344 rats to explore possible mechanisms of adaptation. Of particular interest was ascorbic acid (AA), an antioxidant reported to be protective against O3 injury and found to be increased in O3-adapted rats. Adaptation was induced by exposure to 0.25 ppm O3, 12 hr/day for 6 or 14 weeks and evaluated with a challenge test, one that reexposed rats to 1.0 ppm O3 and measured attenuation in the O3 effect on frequency of breathing. Pulmonary function was assessed 1 day postexposure and adaptation and BALF were evaluated 1, 3, and 7 days postexposure. Results showed that forced vital capacity increased over time but decreased due to exposure and that the 14-week, O3-exposed rats had an increase in forced expiratory flow rate. All of the O3-exposed rats that were tested demonstrated adaptation on Postexposure Days 1, 3, and 7, but it was diminished on Day 7. Adaptation was also more pronounced in rats exposed for 14 weeks. Except for AA, BALF levels of total protein, potassium, lysozyme, uric acid, and alpha-tocopherol were unaffected by O3 exposure. Lactic acid dehydrogenase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, and total glutathione were also assayed but were always below detectable limits. Ascorbic acid concentrations were elevated on Days 1, 3, and 7, showing postexposure patterns similar to those found for adaptation. Significant correlation was found between AA concentration and the magnitude of adaptation (r = 0.91, p < 0.002). We conclude that AA may play an important role in mechanisms associated with O3 adaptation in rats.