Kay M. Crissman

Comparison of Antioxidant Substances in Bronchoalveolar Lavage Cells and Fluid from Humans, Guinea Pigs, and Rats

Antioxidants located in the lining layer of the respiratory tract may be important in determining sensitivity of lung tissues to inhaled pollutants. This study addressed species differences in the amounts of ascorbic acid (AH2), glutathione (GSH), uric acid (UA), and alpha-tocopherol (AT) in bronchoalveolar lavage (BAL) fluid and cells of humans, guinea pigs, and rats. Protein and lipid phosphorus (lipid P) were used as normalizing factors. More than 90% of the lavageable AH2, UA, GSH, protein, and lipid P was present in the extracellular fraction of BAL in rats and guinea pigs, while over 95% of the lavageable AT was located in the BAL cells. BAL fluid AH2/protein in rats was 7- to 9-fold higher than in humans and guinea pigs. However, human BAL fluid had 2- to 8-fold higher UA/protein, GSH/protein, and AT/protein ratios than rats and guinea pigs. In BAL cells, rats had higher AH2/protein and AT/protein ratios than guinea pigs and humans, and both rats and guinea pigs had higher GSH and AT/protein ratios than humans. Individual variability among humans in the BAL fluid and cellular antioxidants was generally greater than in the laboratory animals. These data demonstrate that some large species differences exist in BAL fluid and cellular antioxidants which could affect susceptibility to oxidant pollutants.

Particulate matter in cigarette smoke alters iron homeostasis to produce a biological effect.

RATIONALE Lung injury after cigarette smoking is related to particle retention. Iron accumulates with the deposition of these particles. OBJECTIVES We tested the postulate that (1) injury after smoking correlates with exposure to the particulate fraction of cigarette smoke, (2) these particles alter iron homeostasis, triggering metal accumulation, and (3) this alteration in iron homeostasis affects oxidative stress and inflammation. METHODS Rats and human respiratory epithelial cells were exposed to cigarette smoke, filtered cigarette smoke, and cigarette smoke condensate (the particulate fraction of smoke), and indices of iron homeostasis, oxidative stress, and inflammatory injury were determined. Comparable measures were also evaluated in nonsmokers and smokers. MEASUREMENTS AND MAIN RESULTS After exposure of rats to cigarette smoke, increased lavage concentrations of iron and ferritin, serum ferritin levels, and nonheme iron concentrations in the lung and liver tissue all increased. Lavage ascorbate concentrations were decreased, supporting an oxidative stress. After filtering of the cigarette smoke to remove particles, most of these changes were reversed. Exposure of cultured respiratory epithelial cells to cigarette smoke condensate caused a similar accumulation of iron, metal-dependent oxidative stress, and increased IL-8 release. Lavage samples in healthy smokers and smoking patients with chronic obstructive pulmonary disease revealed elevated concentrations of both iron and ferritin relative to healthy nonsmokers. Lavage ascorbate decreased with cigarette smoking. Serum iron and ferritin levels among smokers were increased, supporting systemic accumulation of this metal after cigarette smoke exposure. CONCLUSIONS We conclude that cigarette smoke particles alter iron homeostasis, both in the lung and systemically.

ASCORBIC ACID IS DECREASED IN INDUCED SPUTUM OF MILD ASTHMATICS

Asthma is primarily an airways inflammatory disease, and the bronchial airways have been shown to be particularly susceptible to oxidant-induced tissue damage. The antioxidant ascorbic acid (AA) plays an essential role in defending against oxidant attack in the airways. Decreased levels of AA have been reported in the plasma and BAL fluid of asthmatics, but not at the site directly proximal to asthma pathology, the bronchial airways. We investigated whether asthmatics have deficient levels of AA in the airways compared to healthy subjects. We performed induced sputum (IS) in a group of mild asthmatics (n = 16) and healthy controls (n = 18) in order to compare constitutive levels of antioxidants in the airways of these two groups. We report that asthmatics had significantly decreased AA in both the cellular (17 +/- 3 ng/10(6) cells vs. 40 +/- 4 ng/10(6) cells) and fluid-phase fraction (616 +/- 152 ng/ml vs. 937 +/- 161 ng/ml) of the IS sample compared to normals. No differences were found with glutathione (GSH) and alpha-tocopherol. These results suggest that AA deficiency may be either an underlying factor in the pathophysiology of asthma or a response to asthmatic airways inflammation.

Ozone-induced tissue injury and changes in antioxidant homeostasis in normal and ascorbate-deficient guinea pigs.

It has been reported previously that ozone (O3) toxicity from acute (4 hr) exposure is enhanced by ascorbate (AH2) deficiency in guinea pigs. We hypothesized that lung injury from continuous 1-week O3 exposure would also be increased under conditions of AH2 deficiency because of (1) a diminished antioxidant pool to counteract the oxidant challenge, (2) impaired reparation of tissue injury, and/or (3) altered antioxidant redox homeostasis. Female Hartley guinea pigs (260-330 g) were made AH2 deficient by providing a diet similar to guinea pig chow, but having no AH2. The dietary regimen was started 1 week prior to exposure and was continued during exposure to O3 (0, 0.2, 0.4, or 0.8 ppm, 23 hr/day, 7 days) as well as 1 week post-exposure. Bronchoalveolar lavage (BAL) and tissue AH2 were measured in subgroups at the beginning of exposure (1 week on the AH2-deficient diet), at its termination and 1 week post-exposure. AH2 measured in ear tissue punches proved to be an easy and effective monitor for AH2 deficiency. One week on the AH2-deficient diet caused a 70-80% drop in ear, lung and liver AH2, while AH2 in BAL was decreased by 90%. Immediately after the exposure, total BAL protein and albumin (markers of lung permeability) were increased (approximately 50%) at 0.8 ppm with no difference between the dietary groups. O3 caused an increase in total BAL cells and neutrophils in a concentration-dependent manner with only a slight augmentation due to diet. Exposure to O3 caused an increase in lung and BAL AH2 in normal guinea pigs. Glutathione and uric acid were also increased in the lung and BAL after O3 exposure (40-570%) in both dietary groups, and the levels remained elevated during the recovery period. Lung alpha-tocopherol was not changed due to O3. A significant overall diet-related decrease was seen in AH2-deficient guinea pigs, immediately after the exposure and recovery. In summary, lung injury/inflammation following 1 week O3 exposure and recovery were minimally affected by AH2 deficiency. Antioxidants also appeared to increase in response to O3 exposure despite the deficiency in AH2.

Biomarkers of Dose and effect of Inhaled Ozone in Resting versus exercising Human subjects: comparison with Resting Rats

To determine the influence of exercise on pulmonary dose of inhaled pollutants, we compared biomarkers of inhaled ozone (O3) dose and toxic effect between exercise levels in humans, and between humans and rats. Resting human subjects were exposed to labeled O3 (18O3, 0.4 ppm, for 2 hours) and alveolar O3 dose measured as the concentration of excess 18O in cells and extracellular material of nasal, bronchial, and bronchoalveolar lavage fluid (BALF). We related O3 dose to effects (changes in BALF protein, LDH, IL-6, and antioxidant substances) measurable in the BALF. A parallel study of resting subjects examined lung function (FEV1) changes following O3. Subjects exposed while resting had 18O concentrations in BALF cells that were 1/5th of those of exercising subjects and directly proportional to the amount of O3 breathed during exposure. Quantitative measures of alveolar O3 dose and toxicity that were observed previously in exercising subjects were greatly reduced or non-observable in O3 exposed resting subjects. Resting rats and resting humans were found to have a similar alveolar O3 dose.

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.

Combination treatment with high-dose vitamin C and alpha-tocopherol does not enhance respiratory-tract lining fluid vitamin C levels in asthmatics.

Oxidative stress plays a significant role in allergic airway inflammation. Supplementation with alpha-tocopherol (alone or combined with ascorbate/vitamin C) has been assessed as an intervention for allergic airway diseases with conflicting results. Enhancing levels of airway antioxidants with oral supplements has been suggested as an intervention to protect individuals from the effect of inhaled oxidants, although it is unclear whether supplementation changes tocopherol or vitamin C levels in both serum and airway fluids. Our objective was to obtain pilot safety and dosing data from 14 allergic asthmatic volunteers examining the effect of daily combination oral therapy with 500 mg alpha-tocopherol (α T) and 2 g vitamin C for 12 wk. We examined serum and airway fluid and cellular levels of alpha- and gamma-tocopherol (γ T) and vitamin C to plan for future studies of these agents in asthma and allergic rhinitis. Six volunteers completed 12 wk of active treatment with α T and vitamin C and 8 completed placebo. Blood and sputum samples were obtained at baseline and at 6 wk and 12 wk of therapy and were analyzed for α T, γ T, and vitamin C levels in the serum, sputum supernatant, and sputum cells. Combination treatment increased serum vitamin C and significantly decreased sputum α T and serum γ T levels. No changes were found in sputum supernatant or sputum cell vitamin C or serum α T levels in the active treatment group. In conclusion, supplementation with α T and high-dose vitamin C does not augment vitamin C levels in the respiratory-tract lining fluid.

Iron homeostasis and oxidative stress in idiopathic pulmonary alveolar proteinosis: a case-control study

BackgroundLung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.MethodsHealthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.ResultsLavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.ConclusionIron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease.

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.

Oxalate deposition on asbestos bodies

We report on a deposition of oxalate crystals on ferruginous bodies after occupational exposure to asbestos demonstrated in 3 patients. We investigated the mechanism and possible significance of this deposition by testing the hypothesis that oxalate generated through nonenzymatic oxidation of ascorbate by asbestos-associated iron accounts for the deposition of the crystal on a ferruginous body. Crocidolite asbestos (1000 microg/mL) was incubated with 500 micromol H(2)O(2) and 500 micromol ascorbate for 24 hours at 22 degrees C. The dependence of oxalate generation on iron-catalyzed oxidant production was tested with the both the metal chelator deferoxamine and the radical scavenger dimethylthiourea. Incubation of crocidolite, H(2)O(2), and ascorbate in vitro generated approximately 42 nmol of oxalate in 24 hours. Oxalate generation was diminished significantly by the inclusion of either deferoxamine or dimethylthiourea in the reaction mixture. Incubation of asbestos bodies and uncoated fibers isolated from human lung with 500 micromol H(2)O(2) and 500 micromol ascorbate for 24 hours at 22 degrees C resulted in the generation of numerous oxalate crystals. We conclude that iron-catalyzed production of oxalate from ascorbate can account for the deposition of this crystal on ferruginous bodies.