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Am J Physiol Lung Cell Mol Physiol 292: L134-L143, 2007. First published August 11, 2006; doi:10.1152/ajplung.00534.2005 Free Article
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Lung injury after ozone exposure is iron dependent

Andrew J. Ghio,1 Jennifer L. Turi,2 Michael C. Madden,1 Lisa A. Dailey,1 Judy D. Richards,1 Jacqueline G. Stonehuerner,1 Daniel L. Morgan,3 Steven Singleton,4 Laura M. Garrick,4 and Michael D. Garrick4

1National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, and 3National Institute of Environmental Health Sciences and National Toxicology Program, Research Triangle Park; 2Department of Pediatrics, Duke University Medical Center, Durham, North Carolina; and 4Department of Biochemistry, State University of New York, Buffalo, New York

Submitted 19 December 2005 ; accepted in final form 1 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We tested the hypothesis that oxidative stress and biological effect after ozone (O3) exposure are dependent on changes in iron homeostasis. After O3 exposure, healthy volunteers demonstrated increased lavage concentrations of iron, transferrin, lactoferrin, and ferritin. In normal rats, alterations of iron metabolism after O3 exposure were immediate and preceded the inflammatory influx. To test for participation of this disruption in iron homeostasis in lung injury following O3 inhalation, we exposed Belgrade rats, which are functionally deficient in divalent metal transporter 1 (DMT1) as a means of iron uptake, and controls to O3. Iron homeostasis was disrupted to a greater extent and the extent of injury was greater in Belgrade rats than in control rats. Nonheme iron and ferritin concentrations were higher in human bronchial epithelial (HBE) cells exposed to O3 than in HBE cells exposed to filtered air. Aldehyde generation and IL-8 release by the HBE cells was also elevated following O3 exposure. Human embryonic kidney (HEK 293) cells with elevated expression of a DMT1 construct were exposed to filtered air and O3. With exposure to O3, elevated DMT1 expression diminished oxidative stress (i.e., aldehyde generation) and IL-8 release. We conclude that iron participates critically in the oxidative stress and biological effects after O3 exposure.

air pollution; lung diseases; ferritin; oxidants; free radicals

OZONE (O3) is a major component of air pollution produced by a reaction between ultraviolet light, hydrocarbons, and nitrogen oxides. In numerous urban areas of the United States, concentrations of O3 are elevated, reflecting vehicular use and the emission of precursors for the generation of this oxidant gas. O3 inhalation results in an influx of inflammatory cells into the lower respiratory tract, airway hyperreactivity, and lung injury in humans (1, 6). Recent epidemiologic investigation also suggests an association between O3 exposure and mortality (2).

Because solubility of O3 in water is poor, O3 does not diffuse into tissues. Rather, O3 reacts with molecules at the air-fluid boundary. The biological effect of O3 is associated with an oxidative stress in the lower respiratory tract (15, 23). O3 will react directly with unsaturated fatty acids to produce hydrogen peroxide and aldehydes (24). Although the exact mechanism that generates this oxidative stress is not well understood, the presence of iron can increase oxidant generation after O3 interaction with aqueous media (5, 13, 27) and produce hydroxyl radicals (23). O3 inhalation significantly alters iron homeostasis in the lower respiratory tract of horses, with resulting elevations in the total and free concentrations of this catalytically active metal (26). The pretreatment of other animal models with the chelator deferoxamine diminishes airway hyperresponsiveness and lung injury after O3 exposure (18, 20), supporting participation of iron in the respiratory consequences of O3 inhalation. Given the association of iron with O3-induced injury, we hypothesized that O3 alters iron homeostasis and elevates the concentration of catalytically active metal to generate an oxidative stress and effect a lung injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human exposure to O3. All protocols and consent forms were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. Volunteers responding to a newspaper advertisement were prescreened over the telephone using the following criteria: they were 18–35 yr of age, were nonsmokers for ≥5 yr before the study, had no history of allergies or respiratory diseases, and were not taking any medication. Each subject (n = 16) was exposed to filtered room air for 2 h while alternately resting and exercising for 15-min periods on a recumbent bicycle (with exercise load adjusted to produce ventilation of 20 l·min–1·m body surface area–2). At 14 days after air exposure, each subject was exposed to 0.4 ppm O3, so each was paired as both an experimental and a control subject. Bronchoscopy with lavage was performed 18 h after exposures to filtered air and O3 (11). Samples were put on ice immediately after aspiration and centrifuged at 600 g for 10 min at 4°C. The supernatant was assayed for iron, transferrin (Tf), Tf receptor (TfR), lactoferrin, and ferritin (see below).

Exposure of Sprague-Dawley rats to O3. The experimental protocols were approved by the Institutional Animal Care and Use Committee at the US Environmental Protection Agency. Sixty-day-old, male Sprague-Dawley rats (Charles River, Raleigh, NC; 250–300 g body wt) were used 1 wk after their arrival. Animals in 0.3-m3 Rochester-type chambers were exposed to filtered air, 2 ppm O3, or 5 ppm O3 for 2 h. At specified times after completion of the exposure, the animals (n = 6 for each exposure at each time point) were anesthetized with halothane. After blood was collected by intracardiac puncture, the aorta was severed to euthanize the animal. Tracheal lavage was carried out with normal saline (35 ml/kg body wt), and the lavage fluid was stored immediately on ice. All concentrations in lavage fluid are expressed per milliliter retrieved. Lung and liver tissue were resected.

Serum and lavage fluid were analyzed for iron, Tf, and ferritin. Lavage lactate dehydrogenase (LDH) and protein concentrations were measured using a commercially available kit (Sigma, St. Louis, MO) and Coomassie Plus protein assay reagent (Pierce, Rockford, IL), respectively, to determine the presence of injury. Nonheme iron concentration was measured in resected lung and liver tissue.

In some animals (n = 4), lungs were inflation fixed with 10% formalin for 24 h and then transferred to 70% ethanol. Antibodies specific for ferritin and divalent metal transporter 1 (DMT1) were used for immunohistochemical staining, as previously described (30).

Exposure of Belgrade rats to O3. Experimental protocols were approved by the Institutional Animal Care and Use Committees at the State University of New York (Buffalo, NY) and the National Institute for Environmental Health Sciences. Belgrade rats are functionally deficient in DMT1 as a means of metal cation transport (5). The rats used in this investigation were N14 generation crossed into a Fischer 344 background (4); thus 86.7% of their genome was Fischer 344. For improvement of the husbandry of these b/b rats, the colony was maintained on an iron-supplemented rat chow (PMI 5001 with addition of 130 ppm FeSO4) (10). Use of N14 +/b littermates as controls eliminated the likelihood that strain differences contributed to the results.

Rats were exposed to filtered air or 0.3 ppm O3 for 24 h (n = 12 per exposure for a total of 24 animals). After the 24-h exposure, the animals were euthanized by intraperitoneal injection of pentobarbital sodium (Nembutal), exsanguinated, and subjected to tracheal lavage. After centrifugation for 10 min at 600 g for removal of cells, protein and LDH concentrations in the lavage fluid were measured to assess injury. Lavage concentrations of iron, Tf, and ferritin were also quantified to evaluate metal homeostasis. All concentrations in lavage fluid are expressed per milliliter retrieved.

Exposure of human bronchial epithelial cells to O3. Human bronchial epithelial (HBE) cells were obtained from healthy, nonsmoking adult volunteers by cytological brushing of the airways during bronchoscopy. These cells were expanded to passage 3 in bronchial epithelial growth medium (Clonetics, San Diego, CA), plated on 0.4-µm-pore collagen-coated filters (Trans-CLR, Costar, Cambridge, MA) at a density of 1 x 105 cells/filter, and transferred to 12-well culture plates. The cells were maintained in a 1:1 mixture of bronchial epithelial growth medium and high glucose DMEM with singlequot supplements, bovine pituitary extracts (13 mg/ml), bovine serum albumin (1.5 g/ml), and nystatin (20 U/ml) in 0.5 ml in the apical chamber and 1.5 ml in the basal chamber. Fresh medium was provided every 48 h. Retinoic acid was added on day 2 for promotion of differentiation, and an air-liquid interface was created on day 6 by removal of the apical medium. The cells were maintained until they had uniformly differentiated into ciliated, mucus-producing cells ~10 days later.

HBE cells were exposed to filtered air or 0.4 ppm O3 for 5 h in in vitro exposure chambers (25); each gas was provided at 20 l/min, balanced with 5% CO2, and at 88% relative humidity. At 24 h after the exposure, a commercially available kit (R & D Systems, Minneapolis, MN) was used to remove the medium for determination of IL-8, and the HBE cells were scraped into 0.5 ml of Hanks’ balanced salt solution and disrupted using a 25-gauge needle. The concentrations of iron, Tf, and ferritin were quantified.

An additional set of cells were treated as described above. The cells were scrapped into 1.0 ml of 2,4-dinitrophenylhydrazine solution (0.125% in acetonitrile) and vortexed. Saline added to 2,4-dinitrophenylhydrazine was used as a blank. Aldehydes (C2–C10) were quantified as an index of oxidative stress (19).

Overexpression of DMT1 in human embryonic kidney cells. DMT1 cDNA was inserted into the Gateway system (Invitrogen, Carlsbad, CA) (17). The construct contained a COOH-terminal FLAG tag (for convenience in identifying transfected cells) following the open reading frame for mouse DMT1, which starts at exon 2 and terminates at exon 17. This system was used to reclone the constructs into pDEST31. Human embryonic kidney (HEK 293-F) cells containing a TetR:Hyg element for beta testing (Invitrogen) were plated on 0.4-µm-pore collagen-coated filters (Trans-CLR, Costar) and transferred to 12-well culture plates. The plates were incubated at 37°C with 5% CO2 in DMEM supplemented with nonessential amino acids and tetracycline-free 10% FBS (Clontech, Palo Alto, CA) containing 200 µg/ml of hygromycin, 100 µg/ml of streptomycin, and 100 U/ml of penicillin (all from Invitrogen). Geneticin (600 µg/ml; G-418, Invitrogen) selection was used to obtain permanent cell lines that contained the empty pDEST31 vector or the same vector with DMT1. The vector pDEST31 has a cytomegalovirus promoter next to a tet-on site. These cell lines were used in experiments after 10–30 passages. Cells were not exposed to doxycycline before any experiment, but DMT1 expression was induced by 50 nM doxycycline for 24 h, except where noted. Concentrations of iron, ferritin, and IL-8 were measured as described for HBE cells. Quantitative RT-PCR was performed (12) with minor changes: RNA was extracted with RNeasy (Qiagen) and converted to cDNA. Quantitative RT-PCR was performed for GAPDH using primers and probe as described above; for mouse –iron response element DMT1, these were as follows: 5'-CGTACCGCCTGGGACTGA-3' (forward), 5'-GTCATCTGGACACCACTGAGTCA-3' (reverse), and 5'-CAGCCTGAACTCTATCTTCTGAACACCGTGG-3' (probe).

The program used for the Bio-Rad iCycler was 2 min at 50°C, 8 min 30 s at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Data were reduced initially using Bio-Rad software and then imported into Stata (Stata, College Station, TX) for quantification.

Western blot for DMT1. Cells were washed with ice-cold PBS, lysed with buffer containing 1% NP-40, 0.5% deoxycholate, and 0.1% SDS and protease inhibitors (Cocktail Set III, Calbiochem, La Jolla, CA), and then sheared through a 22-gauge needle. Protein content was determined using the Bradford assay (Bio-Rad, Hercules, CA). The remainder of the sample was mixed with an equal volume of 4x sample loading buffer (0.5 M Tris·HCl, pH 6.8, 10% glycerol, 2% SDS, 0.7 mM beta-mercaptoethanol, and 0.05% bromphenol blue). Protein samples (50 µg) were separated by electrophoresis on a 4–15% SDS acrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 3% nonfat milk in PBS and incubated with an antibody directed against DMT1 (12). The membrane was stained with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and developed using enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech). For evaluation of a control protein, an equal amount of protein was added to each lane and the blots were also probed with antibody to actin (Santa Cruz Biotechnology).

Nonheme iron concentrations. Concentrations of nonheme iron in serum, lavage fluid, resected lung and liver tissue, and cell extracts were quantified using inductively coupled plasma optical emission spectroscopy (ICPOES) (Optima 4300D, Perkin Elmer, Norwalk, CT) operating at a wavelength of 238.204 nm. To 1.0 ml of serum or lavage fluid, 1.0 ml of 6 N HCl-20% trichloroacetic acid was added, and the specimen was hydrolyzed by heating to 70°C for 18 h. After centrifugation at 1,200 g for 10 min, iron concentrations in the supernatant were measured. In tissues, nonheme iron was determined after addition of 10.0 ml of 3 N HCl-10% trichloroacetic acid per gram of tissue and heating to 70°C for 18 h. After centrifugation at 600 g for 10 min, concentrations of the metal in the supernatant were measured. Finally, cells were scraped into 1.0 ml of 3 N HCl-10% trichoroacetic acid, hydrolyzed, and centrifuged. Iron concentrations in the supernatant were quantified.

Concentrations of Tf, TfR, lactoferrin, and ferritin. Tf protein concentrations in lavage supernatants were analyzed using a commercially available kit, controls, and standards from INCSTAR (Stillwater, MN). All were adapted for use on the Cobas Fara II centrifugal spectrophotometer. TfR and ferritin were measured with commercially available ELISA kits (R & D Systems). Lactoferrin concentrations were also measured using a commercially available ELISA kit (Calbiochem).

Statistics. Values are means ± SE. Differences between multiple groups were compared using one-way analysis of variance. Scheffé’s test was used for post hoc analysis. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
O3 exposure alters iron homeostasis. To define changes in iron homeostasis after O3 inhalation in humans, healthy volunteers were exposed to filtered air and 0.4 ppm O3 for 2 h (with 1 h during exercise). Bronchoscopy performed 18 h after O3 exposure demonstrated disrupted iron metabolism in the lower respiratory tract (Table 1), with elevated concentrations of iron, Tf, TfR, lactoferrin, and ferritin in the lavage fluid after O3 exposure. Protein concentration in this human lavage, however, was not elevated, suggesting that the loss of the alveolar-capillary barrier function after O3 exposure is unlikely to have contributed to these differences (26). There was an influx of neutrophils after O3 inhalation (26), although it is unclear whether these neutrophils contributed to the changes in lavage indexes of iron homeostasis. The lack of elevation in lavage protein concentration among human volunteers exposed to O3 reflects a lower concentration of pollutant (relative to the animal models employed in this investigation).


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  		Table 1. Indexes of iron homeostasis in human bronchoalveolar lavage fluid after exposure to air and O3

 
Changes in iron homeostasis in response to O3 were investigated further using an animal model. Iron concentrations in lavage fluid were significantly elevated immediately after exposure of Sprague-Dawley rats to 2 and 5 ppm O3. This increase persisted for 96 h after exposure to 5 ppm O3, although it returned to baseline at 96 h after exposure to 2 ppm O3 (Fig. 1A). Tf (Fig. 1B) and ferritin (Fig. 1C) in the lavage of the animals similarly increased after O3 exposure at low and high concentrations. Finally, nonheme iron in lung tissue resected from the rats was elevated immediately after O3 exposure, remained elevated for 24 h, and then returned to baseline by 96 h (Fig. 1D). O3-induced expression of ferritin (Fig. 2, A and B) and DMT1 (Fig. 2, C and D) was demonstrated throughout the rat lung by immunohistochemistry, with macrophages and lung epithelial cells exhibiting some elevation in the expression of these storage and transport proteins.


Figure 1
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  		Fig. 1. Iron homeostasis is altered in lungs of rats exposed to O3. Sprague-Dawley rats were exposed to air, 2 ppm O3, or 5 ppm O3 for 2 h. At 0, 24, and 96 h after exposure, the rats were tracheally lavaged, the lung was resected, and indexes of iron homeostasis were measured. A: nonheme iron measured by inductively coupled plasma spectroscopy [ICPOES, wavelength ({lambda}) = 238.204 nm]. B and C: transferrin (Tf) and ferritin in lavage fluid measured by immunoprecipitation and ELISA, respectively. D: lung iron measured by ICPOES. Values are means ± SE (n = 6). *P < 0.05 vs. air.

 

Figure 2
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  		Fig. 2. O3 exposure increases expression of ferritin and DMT1. Sprague-Dawley rats were exposed to air or 2 ppm O3 for 2 h. At 24 h after exposure, the lungs were inflation fixed and sectioned, and ferritin and DMT1 expression was determined by immunohistochemistry. Expression of ferritin (top) and DMT1 (bottom) was increased in rats exposed to O3 (right) relative to those exposed to air (left). Expression of these storage and transport proteins was elevated in macrophages (thin arrows) and lung epithelial cells (thick arrows).

 
Exposure of Sprague-Dawley rats to O3 also immediately altered indexes of systemic iron metabolism; however, some of these changes were the opposite of those in the lower respiratory tract (Fig. 3). Serum iron decreased in rats exposed to O3 over the interval studied, whereas serum Tf initially decreased but then recovered (more quickly for 2 ppm O3). Serum ferritin in the animals increased after O3 exposure, possibly reflecting an increased sequestration of iron; this elevation persisted for 96 h. Liver nonheme iron also increased, but only after 96 h, suggesting long-term storage of sequestered iron.


Figure 3
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  		Fig. 3. Serum indexes of iron homeostasis are altered after exposure to O3. Sprague-Dawley rats were exposed to air, 2 ppm O3, or 5 ppm O3 for 2 h, and blood was collected immediately after exposure and at 24 and 96 h. A: serum iron measured by ICPOES ({lambda} = 238.204 nm). B and C: serum Tf and ferritin measured by immunoprecipitation and ELISA, respectively. D: liver iron measured by ICPOES. Values are means ± SE (n = 6). *P < 0.05 vs. air.

 
These alterations of iron metabolism in the animals were associated with an O3 concentration-dependent lung injury, as demonstrated by increases in lavage concentrations of protein and LDH immediately and 24 h after O3 exposure (Fig. 4). Significantly more injury was demonstrated after exposure to 5 ppm O3 than 2 ppm O3. LDH levels peaked immediately after exposure to 2 and 5 ppm O3 and remained elevated 24 h after exposure. The peak in lavage protein concentration occurred 24 h after exposure to 2 and 5 ppm O3, but to a much greater extent after 5 ppm O3. The protein concentration returned to baseline by 96 h after exposure to 2 ppm O3 but remained somewhat elevated after 5 ppm O3.


Figure 4
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  		Fig. 4. Protein and lactate dehydrogenase (LDH) increase in lung lavage fluid after exposure to O3. Sprague-Dawley rats were exposed to air, 2 ppm O3, or 5 ppm O3 for 2 h and intratracheally lavaged with normal saline immediately after exposure or after 24 or 48 h. Injury was assessed by measurement of protein concentration in lung lavage fluid using Pierce Coomassie Plus protein assay reagent (A) or LDH concentration using a commercially available kit (B). Values are means ± SE (n = 6). *P < 0.05 vs. air.

 
For evaluation of the in vitro response of cultured airway epithelial cells to O3, HBE cells were exposed to filtered air or 0.4 ppm O3 for 5 h. With trypan blue exclusion methodologyas the criterion, no cytotoxicity was associated with O3 exposures. Consistent with in vivo O3 exposure, after 24 h, the cells exposed to O3 exhibited higher nonheme iron (Fig. 5A) and ferritin (Fig. 5B) concentrations. Furthermore, O3 exposure stimulated oxidative stress as measured by aldehyde generation in the HBE cells (Fig. 5C) and increased release of the proinflammatory mediator IL-8 (Fig. 5D), reflecting a relevant biological effect.


Figure 5
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  		Fig. 5. Altered iron homeostasis in human bronchial epithelial (HBE) cells after exposures to O3. HBE cells, grown at the air-liquid interface and differentiated into a mucociliary epithelium, were exposed to filtered air or 0.4 ppm O3 for 5 h. At 24 h after exposure, iron (A) and ferritin (B) were measured by ICPOES ({lambda} = 238.204) and ELISA, respectively. C: lipid peroxides, as evidence of oxidative stress, measured by HPLC. D: IL-8, a proinflammatory mediator, measured by ELISA. Values are means ± SE (n ≥ 3). *P < 0.05 vs. air.

 
Decreased injury after exposure of Belgrade rats to O3. DMT1 is expressed in most tissues and cell types as an integral membrane protein and functions as a transporter of divalent metal cations, including Fe2+. The capacity of DMT1 to transport metal and its ubiquitous expression make it a candidate for transferrin-independent iron uptake. This mode of transport can result in the sequestration of iron in ferritin, where storage would diminish iron-induced reactive oxygen species. DMT1 participates in the transport and detoxification of some metals associated with an air pollution particle to prevent tissue injury (12). Therefore, to examine the relation between O3-induced iron dysregulation and lung injury, we exposed homozygous Belgrade (b/b) rats, which are functionally deficient in DMT1, and littermate (+/b) controls to 0.3 ppm O3 for 24 h. DMT1 may exacerbate injury by internalizing iron, or, conversely, iron internalized by DMT1 may be critical in ameliorating injury. After exposure of Belgrade rats to O3, indexes of iron homeostasis were altered. Although both genotypes exhibited a significant elevation in iron concentrations 24 h after O3 exposures (Fig. 6A), the homozygous Belgrade rats demonstrated a significantly greater increase in Tf concentration (Fig. 6B) and a significantly attenuated increase in ferritin concentration (Fig. 6C) in lavage fluid compared with heterozygous littermate controls. At 24 h after the initiation of O3 exposure, markers of lung injury demonstrated a significantly greater increase in protein (Fig. 7A) and LDH (Fig. 7B) levels in the Belgrade (b/b) rats than in control (+/b) animals, indicating that diminished DMT1 led to greater injury.


Figure 6
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  		Fig. 6. Altered indexes of iron homeostasis after exposure of Belgrade rats, which are functionally deficient in divalent metal transporter 1 (DMT1), to O3. Belgrade rats and control littermates were exposed to filtered air or 0.3 ppm O3 for 24 h and then intratracheally lavaged. A: lavage concentrations of nonheme iron measured by ICPOES ({lambda} = 238.204). B and C: concentrations of Tf and ferritin measured by immunoprecipitation and ELISA, respectively. Values are means ± SE (n = 12). *P < 0.05 vs. air. **P < 0.05 vs. O3-exposed control.

 

Figure 7
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  		Fig. 7. Elevation of protein and LDH concentrations in lavage fluid of O3-exposed Belgrade rats. Belgrade rats and control littermates were exposed to filtered air or 0.3 ppm O3 for 24 h and then intratracheally lavaged. At 24 h after O3 exposure, injury was significant in control (+/b) animals but was even greater in Belgrade (b/b) rats, as reflected by higher lavage concentrations of protein and LDH. *P < 0.05 vs. air. **P < 0.05 vs. O3-exposed control.

 
Decreased oxidative stress and cytokine release after exposure of HEK 293 cells to O3. To further implicate alterations in iron homeostasis in O3-induced cellular injury, we induced an elevated expression of DMT1 by doxycycline treatment in HEK 293 cells grown at the air-liquid interface and then exposed the cells to filtered air or O3 for 5 h. This treatment increases transcription of DMT1, so that mRNA expression increases by 19.7-fold (±12.0-fold as 95% confidence limits, n = 6, P = 0.014) as detected by quantitative RT-PCR. Trypan blue exclusion showed no cytotoxicity associated with exposure of HEK 293 cells to O3. Elevated DMT1 expression in the HEK 293 cells after treatment with doxycycline was verified by Western blot analysis (Fig. 8). Although exposure of the HEK cells to O3 changed levels of iron and iron-related proteins, HEK cells with induced elevation of DMT1 demonstrated a greater elevation of nonheme iron (Fig. 9A) and ferritin (Fig. 9B) after exposure to O3. Furthermore, oxidative stress was present after exposure of control HEK cells to O3 but was ameliorated in cells overexpressing DMT1 (Fig. 9C). The biological effect of O3 was evaluated by measurement of the release of the proinflammatory cytokine IL-8, which demonstrated that although O3 stimulated the increased release of IL-8 into the medium (Fig. 9D) in control cells, induced DMT1 expression abrogated this response.


Figure 8
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  		Fig. 8. Western blot analysis for DMT1 in HEK 293 cells with uninduced and induced expression of DMT1. Lanes 1–3, extracts of cells grown in the absence of doxycycline induction of DMT1; lanes 4–6, extracts of cells grown in the presence of doxycycline induction of DMT1 (A), with the major band at ~90 kDa. HEK 293 cells with uninduced and induced expression of DMT1 was not different from expression of actin (B), with the band at ~35 kDa.

 

Figure 9
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  		Fig. 9. Altered iron homeostasis in HEK 293 cells with induced expression of DMT1 after exposure to air and O3. DMT1 expression in HEK 293 cells was uninduced or induced with doxycycline for 24 h and then exposed to filtered air or 0.4 ppm O3 for 5 h. At 24 h after exposure, iron (A) and ferritin (B) were measured by ICPOES ({lambda} = 238.204 nm) and ELISA, respectively. Iron and ferritin levels were increased after doxycycline-induced elevations in DMT1 expression. Lipid peroxides, as evidence of oxidative stress, were measured by HPLC (C), and IL-8, a proinflammatory mediator, was measured by ELISA (D) in HEK cells with induced DMT1 expression and compared with concentrations in uninduced controls. Values are means ± SE (n ≥ 3). *P < 0.05 vs. air. **P < 0.05 vs. air and elevated DMT1. ***P < 0.05 vs. O3 and normal DMT1.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This investigation demonstrated that exposure to O3 disrupts iron homeostasis in vivo in the lower respiratory tract of humans and rats and in vitro in HBE cells. The lung injury associated with O3 exposure was increased in an animal model deficient in the iron transporter DMT1, which prevents the uptake and subsequent sequestration of iron within ferritin, demonstrating that functional DMT1 is essential for amelioration of O3-induced injury. Conversely, increasing expression of DMT1 mRNA and protein in an inducible cell line decreases the biological effects of O3.

This disruption in metal metabolism after exposure of healthy humans to O3 was reflected by increased iron, Tf, TfR, lactoferrin, and ferritin concentrations in the lavage fluid. Comparable changes in indexes of iron homeostasis in the lower respiratory tract were observed after exposure of rats to O3. In addition, O3 provoked elevated nonheme iron concentrations in the lung. This response was O3 concentration dependent, but, at least in humans, changes in iron homeostasis occurred at lower O3 levels, at which injury did not occur (26). This disruption of metal homeostasis before the O3-induced lung injury suggests that the changes in iron are not secondary to tissue damage but, potentially, may be a primary event in the biological response. The response was also time dependent, with changes in iron homeostasis preceding inflammation in the rat (i.e., lavage and serum iron and ferritin concentrations were altered before any inflammatory incursion into the lower respiratory tract). In this same model, the lung had reestablished normal iron homeostasis by 4 days after the O3 exposure. Finally, exposure of isolated epithelial cells to O3 resulted in disruption of normal iron homeostasis with changes in iron, Tf, and ferritin. These indexes support an immediate accumulation of iron in the lung and respiratory cells after exposure to O3. Our results extend those of a previous investigation in which concentrations of total and free iron were elevated in the bronchoalveolar lavage fluid of horses exposed to 0.5 ppm O3 for 12 h (21). In addition, human exposure to 0.4 ppm O3 for 1 h has also been associated with ferritin in the lavage fluid of four individuals (30). These results suggest that O3 exposure rapidly leads to an accumulation of biologically active iron, with the metal then provoking host responses to sequester it. These responses include alterations in the concentrations of Tf, TfR, lactoferrin, and ferritin.

In cells exposed to O3, accumulation of iron was associated with an oxidative stress (i.e., elevated concentrations of aldehydes reflecting lipid peroxidation). Previous studies have demonstrated an electron paramagnetic resonance spin signal after exposure of acellular lavage fluid to O3 (23). In our investigation, oxidative stress after O3 exposure of respiratory epithelial cells was diminished by induced expression of the iron transporter protein DMT1. This protein increased cellular transport of iron to allow sequestration in a catalytically less reactive state (i.e., intracellular ferritin). These results are also consistent with lung injury after exposure to particulate matter, where DMT1 expression is critical for minimizing the injury (18).

We also report that oxidative stress is associated with the biological effects after O3 exposure. Cells exposed to O3 exhibited altered iron homeostasis, resulting in an accumulation of metal and aldehydes, whereas increased DMT1 expression diminished lipid peroxidation. Cells with induced DMT1 expression also released decreased concentrations of a proinflammatory mediator. The associations of iron homeostasis and DMT1 expression with oxidative stress and IL-8 release after O3 exposures support participation of iron in the injury after exposure to O3. Belgrade rats with defective DMT1 exhibited an increased injury after O3 exposure. Our results increase the understanding of the role of iron in O3 challenge, as demonstrated by previous investigations. In dogs exposed to 3 ppm O3 for 20 min, elevation of airway hyperreactivity was decreased by treatment with allopurinol and deferoxamine (12). Deferoxamine decreased the inflammatory lung injury in Sprague-Dawley rats after 4 h of exposure to 2 ppm O3. This chelator also prevented airway hyperreactivity after exposure of Brown Norway rats to 3 ppm O3 (29). Finally, in HBE cells exposed to O3, colony-forming efficiency decreased and LDH release increased, with deferoxamine protecting against both effects (9).

The systemic response to exposure of the animal to O3 contrasted in some ways with the lung response. Serum iron and Tf decreased, while ferritin increased. These changes are reminiscent of other disruptions of iron metabolism, such as those after exposure to microbial agents, endotoxin, and particles (3, 16). After exposure to these substances or O3, iron homeostasis in the host is disrupted in a manner that appears to increase availability of the metal to catalyze an oxidative stress. The host must limit such availability of iron if the challenge of O3 in the lower respiratory tract is to be controlled. Systemic sources of iron are decreased, as demonstrated by lower concentrations of iron and Tf. The elevation in serum ferritin reflects the host’s attempt to control availability of the metal in the lung and minimize the consequent oxidative stress and injury. Eventually, the iron is transported from the lung to the liver, where its storage presents less of a threat to the host.

There are large individual differences in human responsiveness to O3. This variation in responsiveness is poorly understood but appears to depend on disease status and other exposures (22). Some of this variability in responsiveness to O3 may reflect differences in initial iron homeostasis or in its regulation with disparities in expression of iron transport and storage proteins, as exemplified by our experiments with Belgrade rats and HEK 293 cells inducible for DMT1 expression, where genetically decreasing or increasing DMT1 expression (presumably thus altering iron homeostasis) led to striking increases or decreases, respectively, in damage after O3 exposure.

We conclude that altered iron homeostasis is an early part of the biological effect of O3 in humans, animals, and cells. Iron participates in the oxidative response, biological effect, and lung injury after O3 exposure. We predict that increased expression of proteins involved in maintaining iron homeostasis helps diminish the biological effect of O3 (e.g., elevated expression of DMT1 among smokers could be part of the diminished response to O3) (8, 28). Similarly, as part of the increased expression of DMT1, the host response to metal accumulated after O3 exposure could contribute to the development of tolerance of future O3 or iron challenges.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Ghio, Campus Box 7315, Human Studies Division, US EPA, 104 Mason Farm Rd., Chapel Hill, NC 27599-7315 (e-mail: ghio.andy{at}epa.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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