Iron and zinc homeostases in female rats with physically active and sedentary lifestyles.

To determine the effects of repeated physical activity on iron and zinc homeostases in a living system, we quantified blood and tissue levels of these two metals in sedentary and physically active Long-Evans rats. At post-natal day (PND) 22, female rats were assigned to either a sedentary or an active treatment group (n = 10/group). The physically active rats increased their use of a commercially-constructed stainless steel wire wheel so that, by the end of the study (PND 101), they were running an average of 512.8 ± 31.9 (mean ± standard error) min/night. After euthanization, plasma and aliquots of liver, lung, heart, and gastrocnemius muscle were obtained. Following digestion, non-heme iron and zinc concentrations in plasma and tissues were measured using inductively coupled plasma optical emission spectroscopy. Concentrations of both non-heme iron and zinc in plasma and liver were significantly decreased among the physically active rats relative to the sedentary animals. In the lung, both metals were increased in concentration among the physically active animals but the change in zinc did not reach significance. Similarly, tissue non-heme iron and zinc levels were both increased in heart and muscle from the physically active group. It is concluded that repeated physical activity in an animal model can be associated with a translocation of both iron and zinc from sites of storage (e.g. liver) to tissues with increased metabolism (e.g. the lung, heart, and skeletal muscle).

Diacetyl exposure disrupts iron homeostasis in animals and cells.

OBJECTIVE: Several mechanisms have been proposed for the biological effect of diacetyl. We tested the postulate that animal and cell exposures to diacetyl are associated with a disruption in iron homeostasis. MATERIALS AND METHODS: Male, Sprague-Dawley rats were intratracheally-instilled with either distilled water or diacetyl. Seven days after treatment, animals were euthanized and the lungs removed, fixed, and embedded. Sections were cut and stained for iron, collagen, and ferritin. Human epithelial (BEAS-2B) and monocytic (THP-1) cells were exposed in vitro to ferric ammonium citrate (FAC), diacetyl, and both FAC and diacetyl. Cell non-heme iron concentrations and ferritin levels were quantified using inductively coupled plasma optical emission spectroscopy and an immunoassay respectively. RESULTS: After exposure of animals to diacetyl, there were airway polypoid lesions which stained positively for both iron and the intracellular storage protein ferritin. Trichrome stain showed a deposition of collagen immediately adjacent to accumulated metal following diacetyl exposure. In in vitro cell exposures, FAC increased non-heme iron concentration but co-incubations of FAC and diacetyl elevated levels to significantly greater values. Levels of ferritin were increased with exposures of BEAS-2B and THP-1 cells to FAC but were similarly greater after co-exposure with FAC and diacetyl. CONCLUSIONS: Results of animal and cell studies support a disruption of iron homeostasis by diacetyl. It is proposed that, following internalization, diacetyl complexes intracellular sources of iron. The cell recognizes a loss of its requisite iron to diacetyl and imports greater concentrations of the metal.

A Fulvic Acid-like Substance Participates in the Pro-inflammatory Effects of Cigarette Smoke and Wood Smoke Particles.

We tested the postulates that (1) a fulvic acid (FA)-like substance is included in cigarette smoke and wood smoke particles (WSP) and (2) cell exposure to this substance results in a disruption of iron homeostasis, associated with a deficiency of the metal and an inflammatory response. The fluorescence excitation-emission matrix spectra of the water-soluble components of cigarette smoke condensate and WSP (Cig-WS and Wood-WS) approximated those for the standard reference materials, Suwanee River and Nordic fulvic acids (SRFA and NFA). Fourier transform infrared spectra for the FA fraction of cigarette smoke and WSP (Cig-FA and Wood-FA), SRFA, and NFA also revealed significant similarities (O-H bond in alcohols, phenols, and carboxylates, C═O in ketones, aldehydes, and carboxylates, and a significant carboxylate content). After exposure to Cig-WS and Wood-WS and the FA standards, iron was imported by respiratory epithelial cells, reflecting a functional iron deficiency. The release of pro-inflammatory mediators interleukin (IL)-8 and IL-6 by respiratory epithelial cells also increased following exposures to Cig-WS, Wood-WS, SRFA, and NFA. Co-exposure of the respiratory epithelial cells with iron decreased supernatant concentrations of the ILs relative to exposures to Cig-WS, Wood-WS, SRFA, and NFA alone. It is concluded that (1) a FA-like substance is included in cigarette smoke and WSP and (2) respiratory epithelial cell exposure to this substance results in a disruption of iron homeostasis associated with both a cell deficiency of the metal and an inflammatory response.

Ozone Reacts With Carbon Black to Produce a Fulvic Acid-Like Substance and Increase an Inflammatory Effect.

Exposure to ambient ozone has been associated with increased human mortality. Ozone exposure can introduce oxygen-containing functional groups in particulate matter (PM) effecting a greater capacity of the particle for metal complexation and inflammatory effect. We tested the postulate that (1) a fulvic acid-like substance can be produced through a reaction of a carbonaceous particle with high concentrations of ozone and (2) such a fulvic acid-like substance included in the PM can initiate inflammatory effects following exposure of respiratory epithelial (BEAS-2B) cells and an animal model (male Wistar Kyoto rats). Carbon black (CB) was exposed for 72 hours to either filtered air (CB-Air) or approximately 100 ppm ozone (CB-O3). Carbon black exposure to high levels of ozone produced water-soluble, fluorescent organic material. Iron import by BEAS-2B cells at 4 and 24 hours was not induced by incubations with CB-Air but was increased following coexposures of CB-O3 with ferric ammonium citrate. In contrast to CB-Air, exposure of BEAS-2B cells and rats to CB-O3 for 24 hours increased expression of pro-inflammatory cytokines and lung injury, respectively. It is concluded that inflammatory effects of carbonaceous particles on cells can potentially result from (1) an inclusion of a fulvic acid-like substance after reaction with ozone and (2) changes in iron homeostasis following such exposure.

Oleic acid and derivatives affect human endothelial cell mitochondrial function and vasoactive mediator production.

BACKGROUND: Inhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed. RESULTS: Dose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F2α, a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression. CONCLUSIONS: Together, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Quartz Disrupts Iron Homeostasis in Alveolar Macrophages To Impact a Pro-Inflammatory Effect.

The biological response of bronchial epithelial cells to particles is associated with a sequestration of cell metal by the particle surface and a subsequent disruption in host iron homeostasis. The macrophage is the cell type resident in the respiratory tract that is most likely to make initial contact with inhaled particles. We tested the postulates that (1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs); and (2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects. Human AMs (1.0 × 106/mL) demonstrated an increased import of iron following particle exposure with nonheme iron concentrations of 0.57 ± 0.03, 1.72 ± 0.09, 0.88 ± 0.09, and 3.21 ± 0.11 ppm in cells exposed for 4 h to media, 500 µM ferric ammonium citrate (FAC), 100 µg/mL silica, and both silica and FAC, respectively. Intracellular ferritin concentrations and iron release were similarly increased after AM exposure to FAC and silica. Silica increased oxidant generation by AMs measured using both dichlorofluorescein diacetate fluorescence and reduction of nitroblue tetrazolium salt. Concentrations of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α in macrophage supernatant increased following 100 µg/mL silica exposure for 24 h. Treatment of AMs with 500 µM FAC decreased both oxidant generation and cytokine release associated with silica exposure, supporting a dependence of these effects on sequestration of cell metal by the particle surface. We conclude that (1) silica exposure disrupts iron homeostasis resulting in increased import, accumulation, and release of the metal; and (2) the altered iron homeostasis following silica exposure impacts oxidant generation and pro-inflammatory effects.

The toxicology of air pollution predicts its epidemiology.

The epidemiologic investigation has successively delineated associations of air pollution exposure with non-malignant and malignant lung disease, cardiovascular disease, cerebrovascular disease, pregnancy outcomes, perinatal effects and other extra-pulmonary disease including diabetes. Defining these relationships between air pollution exposure and human health closely parallels results of an earlier epidemiologic investigation into cigarette smoking and environmental tobacco smoke (ETS), two other particle-related exposures. Humic-like substances (HULIS) have been identified as a chemical component common to cigarette smoke and air pollution particles. Toxicology studies provide evidence that a disruption of iron homeostasis with sequestration of host metal by HULIS is a fundamental mechanistic pathway through which biological effects are initiated by cigarette smoke and air pollution particles. As a result of a common chemical component and a shared mechanistic pathway, it should be possible to extrapolate from the epidemiology of cigarette smoking and ETS to predict associations of air pollution exposure with human disease, which are currently unrecognized. Accordingly, it is anticipated that the forthcoming epidemiologic investigation will demonstrate relationships of air pollution with COPD causation, peripheral vascular disease, hypertension, renal disease, digestive disease, loss of bone mass/risk of fractures, dental disease, eye disease, fertility problems, and extrapulmonary malignancies.

Air pollution particles and iron homeostasis.

BACKGROUND: The mechanism underlying biological effects, including pro-inflammatory outcomes, of particles deposited in the lung has not been defined. MAJOR CONCLUSIONS: A disruption in iron homeostasis follows exposure of cells to all particulate matter including air pollution particles. Following endocytosis, functional groups at the surface of retained particle complex iron available in the cell. In response to a reduction in concentrations of requisite iron, a functional deficiency can result intracellularly. Superoxide production by the cell exposed to a particle increases ferrireduction which facilitates import of iron with the objective being the reversal of the metal deficiency. Failure to resolve the functional iron deficiency following cell exposure to particles activates kinases and transcription factors resulting in a release of inflammatory mediators and inflammation. Tissue injury is the end product of this disruption in iron homeostasis initiated by the particle exposure. Elevation of available iron to the cell precludes deficiency of the metal and either diminishes or eliminates biological effects. GENERAL SIGNIFICANCE: Recognition of the pathway for biological effects after particle exposure to involve a functional deficiency of iron suggests novel therapies such as metal supplementation (e.g. inhaled and oral). In addition, the demonstration of a shared mechanism of biological effects allows understanding the common clinical, physiological, and pathological presentation following exposure to disparate particles. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

The biological effect of asbestos exposure is dependent on changes in iron homeostasis.

Functional groups on the surface of fibrous silicates can complex iron. We tested the postulate that (1) asbestos complexes and sequesters host cell iron resulting in a disruption of metal homeostasis and (2) this loss of essential metal results in an oxidative stress and biological effect in respiratory epithelial cells. Exposure of BEAS-2B cells to 50 µg/mL chrysotile resulted in diminished concentrations of mitochondrial iron. Preincubation of these cells with 200 µM ferric ammonium citrate (FAC) prevented significant mitochondrial iron loss following the same exposure. The host response to chrysotile included increased expression of the importer divalent metal transporter-1 (DMT1) supporting a functional iron deficiency. Incubation of BEAS-2B cells with both 200 µM FAC and 50 µg/mL chrysotile was associated with a greater cell accumulation of iron relative to either iron or chrysotile alone reflecting increased import to correct metal deficiency immediately following fiber exposure. Cellular oxidant generation was elevated after chrysotile exposure and this signal was diminished by co-incubation with 200 µM FAC. Similarly, exposure of BEAS-2B cells to 50 µg/mL chrysotile was associated with release of the proinflammatory mediators interleukin (IL)-6 and IL-8, and these changes were diminished by co-incubation with 200 µM FAC. We conclude that (1) the biological response following exposure to chrysotile is associated with complexation and sequestration of cell iron and (2) increasing available iron in the cell diminished the effects of asbestos exposure.

Wood Smoke Particle Sequesters Cell Iron to Impact a Biological Effect.

The biological effect of an inorganic particle (i.e., silica) can be associated with a disruption in cell iron homeostasis. Organic compounds included in particles originating from combustion processes can also complex sources of host cell iron to disrupt metal homeostasis. We tested the postulate that (1) wood smoke particle (WSP) sequesters host cell iron resulting in a disruption of metal homeostasis, (2) this loss of essential metal results in both an oxidative stress and biological effect in respiratory epithelial cells, and (3) humic-like substances (HULIS), a component of WSP, have a capacity to appropriate cell iron and initiate a biological effect. BEAS-2B cells exposed to WSP resulted in diminished concentrations of mitochondrial (57)Fe, whereas preincubation with ferric ammonium citrate (FAC) prevented significant mitochondrial iron loss after such exposure. Cellular oxidant generation was increased after WSP exposure, but this signal was diminished by coincubation with FAC. Similarly, exposure of BEAS-2B cells to 100 µg/mL WSP activated mitogen-activated protein (MAP) kinases, elevated NF-E2-related factor 2/antioxidant responsive element (Nrf2 ARE) expression, and provoked interleukin (IL)-6 and IL-8 release, but all these changes were diminished by coincubation with FAC. The biological response to WSP was reproduced by exposure to 100 µg/mL humic acid, a polyphenol comparable to HULIS included in the WSP that complexes iron. We conclude that (1) the biological response following exposure to WSP is associated with sequestration of cell iron by the particle, (2) increasing available iron in the cell diminished the biological effects after particle exposure, and (3) HULIS included in WSP can sequester the metal initiating the cell response.

Particle retention by respiratory epithelial cells is associated with persistent biological effect.

The biological effect of particles on respiratory epithelial cells involves, in part, the generation of an oxidative stress and a consequent cascade of reactions culminating in inflammatory mediator release. Whether there is either an immediate, transitory activation or a persistent response of the cells to the particles has not been established. We tested the postulate that respiratory epithelial cells exposed to wood smoke particle (WSP) would demonstrate increased oxidative stress and mediator release following re-seeding and propagation of the cells for two generations post-initial exposure. BEAS-2B cells grown to confluence (G0) in 75 cm(2) flasks were treated for 18 h with the WSP at 0, 25, 50 and 100 µg/ml. The flasks were then used to seed another set of flasks as well as 12- and 96-well plates (G1). These flasks were similarly grown to confluence and the process repeated (G2). Cell viability was assayed using trypan blue dye exclusion and was >85%. Dichlorohydrofluorescein fluorescence after exposure of BEAS-2B cells to 50 and 100 µg/ml WSP increased in all three generations when expressed as a ratio to unexposed cells. Similarly, IL-6 and IL-8 release following the initial exposure of cells to 100 µg/ml WSP increased in all three generations when expressed as a ratio to unexposed cells. The persistence of oxidative stress and inflammatory mediator release for two generations of cells beyond the initial exposure supports a postulate of continued cell response to retained particle.

Iron decreases biological effects of ozone exposure.

CONTEXT: Ozone (O3) exposure is associated with a disruption of iron homeostasis and increased availability of this metal which potentially contributes to an oxidative stress and biological effects. OBJECTIVE: We tested the postulate that increased concentrations of iron in cells, an animal model and human subjects would significantly impact the biological effects of O3 exposure. RESULTS: Exposure to 0.4 ppm O3 for 5 h increased mRNA for both Superoxide Dismutase-1 (SOD1) and Cyclooxygenase-2 (COX2) in normal human bronchial epithelial (NHBE) cells. Pre-treatment of NHBE cells with 200 µM ferric ammonium citrate (FAC) for 4 h diminished changes in both SOD1 and COX2 following O3 exposure. mRNA transcript levels and associated protein release of the pro-inflammatory mediators IL-6 and IL-8 were increased by O3 exposure of NHBE cells; changes in these endpoints after O3 exposure were significantly decreased by FAC pre-treatment of the cells. Exposure of CD-1 mice to 2 ppm O3 for 3 h significantly increased lavage indices of inflammation and airflow limitation. Pre-treatment of the animals with pharyngeal aspiration of FAC diminished the same endpoints. Finally, the mean loss of pulmonary function in 19 healthy volunteers exposed to 0.3 ppm O3 for 2 h demonstrated significant correlations with either their pre-exposure plasma ferritin or iron concentrations. DISCUSSION AND CONCLUSION: We conclude that greater availability of iron after O3 exposure does not augment biological effects. On the contrary, increased available iron decreases the biological effects of O3 exposure in cells, animals and humans.

Host and environmental factors affect pulmonary responses measured in bronchoalveolar lavage.

CONTEXT: Bronchoscopy with bronchoalveolar lavage (BAL) is used to measure pulmonary effects in inhalational exposure studies. OBJECTIVES: To determine how host and background environmental factors may affect pulmonary responses in BAL. MATERIALS AND METHODS: We retrospectively analyzed 77 healthy non-smoking volunteers (38 males and 39 females, age 18-35) who participated in a bronchoscopy study to donate cells for in vitro studies. BAL was performed by lavaging one subsegment of both the lingular segment of the left upper lobe and the right middle lobe with 250 ml of sterile normal saline each. We obtained temperature, relative humidity, ambient O3, PM2.5 and PM10 levels from monitor stations in Durham area in North Carolina. We correlated concentrations of leptin, adiponectin, monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-8, ferritin and total lavaged cells in BAL samples with body mass index (BMI), age, ambient O3, PM2.5, PM10, temperature and relative humidity. RESULTS: Increased BMI was associated with higher lavage leptin. Males had higher MCP-1 and total lavaged cells than females. Average PM2.5, PM10 and O3 concentrations before bronchoscopy were 13.7 µg/m(3), 21.2 µg/m(3) and 0.029 ppm, respectively. Using stepwise multiple linear regression, we found positive associations of MCP-1 with BMI, and of total lavaged cells with humidity and O3. There were inverse associations of IL-8 and total lavaged cells with temperature. DISCUSSION AND CONCLUSIONS: Background environmental and host factors may affect some pulmonary responses to ambient pollutants. Interpretation of pulmonary effects in inhalational exposure studies may need to consider the effects of some host and environmental factors.

Biological effects of desert dust in respiratory epithelial cells and a murine model.

As a result of the challenge of recent dust storms to public health, we tested the postulate that desert dust collected in the southwestern United States imparts a biological effect in respiratory epithelial cells and an animal model. Two samples of surface sediment were collected from separate dust sources in northeastern Arizona. Analysis of the PM20 fraction demonstrated that the majority of both dust samples were quartz and clay minerals (total SiO2 of 52 and 57%). Using respiratory epithelial and monocytic cell lines, the two desert dusts increased oxidant generation, measured by Amplex Red fluorescence, along with carbon black (a control particle), silica, and NIST 1649 (an ambient air pollution particle). Cell oxidant generation was greatest following exposures to silica and the desert dusts. Similarly, changes in RNA for superoxide dismutase-1, heme oxygenase-1, and cyclooxygenase-2 were also greatest after silica and the desert dusts supporting an oxidative stress after cell exposure. Silica, desert dusts, and the ambient air pollution particle NIST 1649 demonstrated a capacity to activate the p38 and ERK1/2 pathways and release pro-inflammatory mediators. Mice, instilled with the same particles, showed the greatest lavage concentrations of pro-inflammatory mediators, neutrophils, and lung injury following silica and desert dusts. We conclude that, comparable to other particles, desert dusts have a capacity to (1) influence oxidative stress and release of pro-inflammatory mediators in respiratory epithelial cells and (2) provoke an inflammatory injury in the lower respiratory tract of an animal model. The biological effects of desert dusts approximated those of silica.

Sequestration of mitochondrial iron by silica particle initiates a biological effect.

Inhalation of particulate matter has presented a challenge to human health for thousands of years. The underlying mechanism for biological effect following particle exposure is incompletely understood. We tested the postulate that particle sequestration of cell and mitochondrial iron is a pivotal event mediating oxidant generation and biological effect. In vitro exposure of human bronchial epithelial cells to silica reduced intracellular iron, which resulted in increases in both the importer divalent metal transporter 1 expression and metal uptake. Diminished mitochondrial (57)Fe concentrations following silica exposure confirmed particle sequestration of cell iron. Preincubation of cells with excess ferric ammonium citrate increased cell, nuclear, and mitochondrial metal concentrations and prevented significant iron loss from mitochondria following silica exposure. Cell and mitochondrial oxidant generation increased after silica incubation, but pretreatment with iron diminished this generation of reactive oxygen species. Silica exposure activated MAP kinases (ERK and p38) and altered the expression of transcription factors (nF-κB and NF-E2-related factor 2), proinflammatory cytokines (interleukin-8 and -6), and apoptotic proteins. All of these changes in indexes of biological effect were either diminished or inhibited by cell pretreatment with iron. Finally, percentage of neutrophils and total protein concentrations in an animal model instilled with silica were decreased by concurrent exposure to iron. We conclude that an initiating event in the response to particulate matter is a sequestration of cell and mitochondrial iron by endocytosed particle. The resultant oxidative stress and biological response after particle exposure are either diminished or inhibited by increasing the cell iron concentration.

Growth of human bronchial epithelial cells at an air-liquid interface alters the response to particle exposure.

BACKGROUND: We tested the hypothesis that normal human bronchial epithelial (NHBE) cells 1) grown submerged in media and 2) allowed to differentiate at air-liquid interface (ALI) demonstrate disparities in the response to particle exposure. RESULTS: Following exposure of submerged NHBE cells to ambient air pollution particle collected in Chapel Hill, NC, RNA for IL-8, IL-6, heme oxygenase 1 (HOX1) and cyclooxygenase 2 (COX2) increased. The same cells allowed to differentiate over 3, 10, and 21 days at ALI demonstrated no such changes following particle exposure. Similarly, BEAS-2B cells grown submerged in media demonstrated a significant increase in IL-8 and HOX1 RNA after exposure to NIST 1648 particle relative to the same cells exposed after growth at ALI. Subsequently, it was not possible to attribute the observed decreases in the response of NHBE cells to differentiation alone since BEAS-2B cells, which do not differentiate, showed similar changes when grown at ALI. With no exposure to particles, differentiation of NHBE cells at ALI over 3 to 21 days demonstrated significant decrements in baseline levels of RNA for the same proteins (i.e. IL-8, IL-6, HOX1, and COX2). With no exposure to particles, BEAS-2B cells grown at ALI showed comparable changes in RNA for IL-8 and HOX1. After the same particle exposure, NHBE cells grown at ALI on a transwell in 95% N2-5% CO2 and exposed to NIST 1648 particle demonstrated significantly greater changes in IL-8 and HOX1 relative to cells grown in 95% air-5% CO2. CONCLUSIONS: We conclude that growth of NHBE cells at ALI is associated with a diminished biological effect following particle exposure relative to cells submerged in media. This decreased response showed an association with increased oxygen availability.

Mucus increases cell iron uptake to impact the release of pro-inflammatory mediators after particle exposure.

We tested the hypothesis that (1) mucus production can be included in the cell response to iron deficiency; (2) mucus binds iron and increases cell metal uptake; and subsequently (3) mucus impacts the inflammatory response to particle exposure. Using quantitative PCR, RNA for both MUC5B and MUC5AC in normal human bronchial epithelial (NHBE) cells decreased following exposures to ferric ammonium citrate (FAC). Incubation of mucus-containing material collected from the apical surface of NHBE cells grown at air-liquid interface (NHBE-MUC) and a commercially available mucin from porcine stomach (PORC-MUC) with iron demonstrated an in vitro capacity to bind metal. Inclusion of either NHBE-MUC or PORC-MUC in incubations of both BEAS-2B cells and THP1 cells increased iron uptake. Exposure to sugar acids (N-acetyl neuraminic acid, sodium alginate, sodium guluronate, and sodium hyaluronate) similarly increased cell iron uptake. Finally, increased metal transport associated with mucus was associated with a decreased release of interleukin-6 and -8, an anti-inflammatory effect, following silica exposure. We conclude that mucus production can be involved in the response to a functional iron deficiency following particle exposure and mucus can bind metal, increase cell uptake to subsequently diminish or reverse a functional iron deficiency and inflammatory response following particle exposure.

Disruption of iron homeostasis in mesothelial cells after talc pleurodesis.

The mechanism for biological effects after exposure to particles is incompletely understood. One postulate proposed to explain biological effects after exposure to particles involves altered iron homeostasis in the host. The fibro-inflammatory properties of mineral oxide particles are exploited therapeutically with the instillation of massive quantities of talc into the pleural space, to provide sclerosis. We tested the postulates that (1) in vitro exposure to talc induces a disruption in iron homeostasis, oxidative stress, and a biological effect, and (2) talc pleurodesis in humans alters iron homeostasis. In vitro exposures of both mesothelial and airway epithelial cells to 100 µg/ml talc significantly increased iron importation and concentrations of the storage protein ferritin. Using dichlorodihydrofluorescein, exposure to talc was associated with a time-dependent and concentration-dependent generation of oxidants in both cell types. The expression of proinflammatory mediators was also increased after in vitro exposures of mesothelial and airway epithelial cells to talc. Relative to control lung tissue, lung tissue from patients treated with sclerodesis demonstrated an accumulation of iron and increased expression of iron-related proteins, including ferritin, the importer divalent metal transport-1 and the exporter ferroportin-1. Talc was also observed to translocate to the parenchyma, and changes in iron homeostasis were focally distributed to sites of retention. We conclude that exposure to talc disrupts iron homeostasis, is associated with oxidative stress, and results in a biological effect (i.e., a fibro-inflammatory response). Talc pleurodesis can function as a model of the human response to mineral oxide particle exposure, albeit a massive one.

Exposure to wood smoke particles produces inflammation in healthy volunteers.

OBJECTIVES: Human exposure to wood smoke particles (WSP) impacts on human health through changes in indoor air quality, exposures from wild fires, burning of biomass and air pollution. This investigation tested the postulate that healthy volunteers exposed to WSP would demonstrate evidence of both pulmonary and systemic inflammation. METHODS: Ten volunteers were exposed to filtered air and, 3 weeks or more later, WSP. Each exposure included alternating 15 min of exercise and 15 min of rest for a total duration of 2 h. Wood smoke was generated by heating an oak log on an electric element and then delivered to the exposure chamber. Endpoints measured in the volunteers included symptoms, pulmonary function tests, measures of heart rate variability and repolarisation, blood indices and analysis of cells and fluid obtained during bronchoalveolar lavage. RESULTS: Mean particle mass for the 10 exposures to air and WSP was measured using the mass of particles collected on filters and found to be below the detectable limit and 485±84 µg/m(3), respectively (mean±SD). There was no change in either symptom prevalence or pulmonary function with exposure to WSP. At 20 h after wood smoke exposure, blood tests demonstrated an increased percentage of neutrophils, and bronchial and bronchoalveolar lavage revealed a neutrophilic influx. CONCLUSIONS: We conclude that exposure of healthy volunteers to WSP may be associated with evidence of both systemic and pulmonary inflammation.

Pollutant particles induce arginase II in human bronchial epithelial cells.

Exposure to particulate matter (PM) is associated with adverse pulmonary effects, including induction and exacerbation of asthma. Recently arginase was shown to play an important role in the pathogenesis of asthma. In this study, it was postulated that PM exposure might induce arginase. Human bronchial epithelial cells (HBEC) obtained from normal individuals by endobronchial brushings cultured on an air-liquid interface were incubated with fine Chapel Hill particles (PM2.5, 100 µg/ml) for up to 72 h. Arginase activity, protein expression, and mRNA of arginase I and arginase II were measured. PM2.5 increased arginase activity in a time-dependent manner. The rise was primarily due to upregulation of arginase II. PD153035 (10 µM), an epidermal growth factor (EGF) receptor antagonist, attenuated the PM2.5-induced elevation in arginase activity and arginase II expression. Treatment of HBEC with human EGF increased arginase activity and arginase II expression. Pretreatment with catalase (200 U/ml), superoxide dismutase (100 U/ml), or apocynin (5 µg/ml), an NAD(P)H oxidase inhibitor, did not markedly affect arginase II expression. Treatment of HBEC with arginase II siRNA inhibited the expression of arginase II by 60% and increased IL-8 release induced by PM2.5. These results indicate that PM exposure upregulates arginase II activity and expression in human bronchial epithelial cells, in part via EGF-dependent mechanisms independent of oxidative stress. The elevated arginase II activity and expression may be a mechanism underlying adverse effects induced by PM exposure in asthma patients.