American Journal of Respiratory Cell and Molecular Biology

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.

This research concerns the disruption in iron homeostasis that occurs in the pleura and lungs of patients treated with talc pleurodesis. The accumulation of this metal, the accompanying oxidative stress, and inflammatory events after exposure to talc are comparable to those with other forms of particulate matter. Pleurodesis can function as a model of particle-related biological effect.

Humans are routinely exposed to the particulate matter (PM) included in air pollution, cigarette smoke, environmental tobacco smoke, forest fires, gas and wood stoves, and the burning of biomass other than wood, as well as those particles contacted during the mining and processing of coal and mineral oxides. Many of the major global causes of death reported by the World Health Organization (http://www.who.int/mediacentre/factsheets/fs310/en/index.html) are related to particle exposure, and contact with PM will increase human morbidity and mortality. This particle-related morbidity and mortality include lower respiratory infections, chronic obstructive pulmonary disease, respiratory cancers, coronary heart disease, stroke, and other cerebrovascular diseases (13).

The production of reactive oxygen species (ROS) is fundamental to the biological effect of PM (4), but the specific mechanism of both the generation of oxidants and its relationship with the biological effect after exposure to particles is incompletely understood. One postulate to explain the biological effect after exposure to particles involves altered iron homeostasis in the host after exposure. Exposure to particles introduces a solid–liquid interface into cells. Oxygen-containing functional groups on the PM surface provide the capacity to complex cations, and as a result of its high affinity for oxygen-donor ligands, iron is frequently preferred. Such functional groups can include alcohols, aldehydes, and carboxylates on incompletely combusted carbon (e.g., cigarette smoke, diesel exhaust, and ambient air pollution particles), and silanol groups on silica and silicates. Particles retained in the lung consistently demonstrate a capacity to disrupt iron homeostasis and accumulate host metal (5). Endpoints reflecting oxidative stress and a biological effect can be correlated with this accumulation of iron that follows in vivo exposure to particles (6).

Pathologic processes observed in lungs among individuals exposed to particles include both inflammation and fibrosis (4, 7). In patients with recurrent pleural effusions, the fibro-inflammatory properties of mineral oxide particles are exploited therapeutically with the instillation of massive quantities (i.e., grams) of talc into the pleural space to provide sclerosis (8). This therapeutic use affords an opportunity to examine human tissue for evidence of a disruption in iron homeostasis after an extreme exposure to mineral oxide particles. We accordingly tested the postulates that (1) in vitro exposure to talc induces a disruption of iron homeostasis, oxidative stress, and a biological effect, and (2) talc pleurodesis in humans is also associated with altered iron homeostasis and an accumulation of metal.

Iron Uptake in an Acellular Environment

Talc (Sclerosol, Bryan Corp., Woburn, MA) was agitated in either H2O or 1,000 μM ferric ammonium citrate for 1 hour, centrifuged, and washed with H2O (designated talc and talc–Fe, respectively). Ionizable metal concentrations associated with talc and talc–Fe were measured using inductively coupled plasma optical emission spectroscopy (ICPOES; Model Optima 4300DV; Perkin Elmer, Norwalk, CT).

Generation of Acellular Oxidants

The generation of acellular oxidants by talc and talc–Fe particles was measured with the thiobarbituric acid–reactive products of deoxyribose (9).

Cell Culture

Mesothelial cells (MeT-5A; American Type Culture Collection, Manassas, VA) were cultured in complete growth medium. In addition, BEAS-2B cells on uncoated, plastic 12-well plates in keratinocyte growth medium (Lonza, Walkersville, MD) were used.

RT-PCR

Mesothelial and BEAS-2B cells were exposed to either medium alone or 100 μg/ml talc. Quantitative PCR was performed using Taqman polymerase, with detection on an ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA).

Cell Iron Homeostasis

Mesothelial and BEAS-2B cells were exposed for 4 hours to 200 μM ferric ammonium citrate (FAC), 100 μg/ml talc, and both FAC and talc. Cells were then scraped into 1.0 ml 3 N HCl/10% trichloroacetic acid. After hydrolysis, concentrations of iron and zinc were determined using ICPOES. Cell incubations were repeated for 24 hours, scraped into 0.5 ml PBS, and disrupted, and ferritin concentrations in the lysates were measured using an enzyme immunoassay (Microgenics Corp., Concord, CA).

Generation of Cellular Oxidants

Mesothelial and BEAS-2B cells were grown to confluence in 96-well, white-walled, tissue culture–treated plates (CoStar, Lowell, MA). We loaded 2′7′ dichlorodihydrofluorescein (DCF; 20 μM) diacetate (Sigma Chemical Co., St. Louis, MO), baseline readings were taken on a Perkin Elmer HTS 7000 fluorimeter using 485-nm excitation/535-nm emission filters, and PBS with and without talc was added. Fluorescence was measured at 0, 60, 120, and 180 minutes after addition.

Cellular Release of IL-8 and IL-6

Cells were exposed to either medium or 100 μg/ml talc in medium for 24 hours. Concentrations of IL-8 and IL-6 in the cell medium were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN).

Histochemistry and Immunohistochemisty

Our protocol was approved by the Institutional Review Board of the Duke University Health System. Staining was performed on six surgical specimens collected from patients who (1) had manifested talc pleurodesis 2–15 months previously (mean ± SD, 7 ± 6 months), followed by (2) an extrapleural pneumonectomy. Sections were stained for iron, using Perl's Prussian blue (Sigma). Immunohistochemistry for ferritin was performed using an human anti-ferritin antibody (Dako, Carpinteria, CA) at a dilution of 1:100 (10) and antibodies to divalent metal transport 1 (DMT1) and ferroportin 1 (FPN1) at a dilution of 1:200 (11, 12). Control lung tissue was obtained from (1) patients who had undergone a pneumonectomy for lung cancer, and (2) individuals diagnosed with idiopathic pulmonary fibrosis (IPF) and undergoing an autopsy.

Statistical Analysis

Data are expressed as mean values ± standard errors, unless specified otherwise. Differences between multiple groups were compared using ANOVA.

Scanning electron microscopy of the talc showed significant variability in PM size, with diameters of individual particles ranging from less than 10 μm to greater than 50 μm (Figure 1A). However, the majority of particles had diameters between 10 and 50 μm. No fibers were evident in the talc sample. Energy-dispersive X-ray spectroscopy revealed two peaks consistent with magnesium and silicon (Figure 1B). This finding is in agreement with the ideal molecular formula of talc (Mg3Si4O10(OH)2).

Comparable to numerous silicates (13), talc demonstrated a capacity for iron uptake in an acellular environment. Concentrations of ionizable iron were measured at 0.46 ± 0.11 ppm and 6.48 ± 1.08 ppm, respectively, for talc and talc–Fe, whereas concentrations of ionizable zinc were measured at 0.03 ± 0.01 and 0.01 ± 0.00 ppm, respectively. The capacity of particles to support the in vitro generation of oxidants in an acellular environment was significantly affected by the concentration of associated iron, with talc–Fe producing a significantly greater signal for malondialdehyde relative to talc (Figure 1C). This generation of oxidants was inhibited by the inclusion of either 1,000 μM deferoxamine, a metal chelator, or 1,000 μM dimethylthiourea, a hydroxyl radical scavenger (Figure 1C).

The in vitro exposure of mesothelial cells to talc for 4 hours resulted in an accumulation of nonheme iron. Cell zinc concentrations did not change (Figure 2A). The exposure to FAC alone confirmed the ability of mesothelial cells to import iron (Figure 2A). Co-incubation with both talc and FAC was associated with a greater accumulation of iron relative to FAC exposure alone (Figure 2A). BEAS-2B cells exposed to talc and FAC revealed similar increases in cell nonheme metal. Again, co-incubations revealed an increased cell iron import relative to either talc or FAC alone (Figure 2B). These results demonstrate that the capacity of talc to complex iron in an acellular environment is retained during in vitro mesothelial and airway epithelial cell exposures. In addition, these data suggest that talc could deplete intracellular sources of iron, and resulting in the importation of greater quantities of metal. Comparable to cell nonheme iron concentration, an increase in cell ferritin occurred after exposures of mesothelial and airway epithelial cells to talc, and this increase was even greater after exposures to both FAC and talc (Figure 2C). RNA for ferritin, DMT1 (a major iron importer), and FPN1 (a major iron exporter) did not significantly change 4 hours after exposure of mesothelial cells to 100 μg/ml talc (Table E1 in the online supplement).

A fluorescence method using DCF diacetate demonstrated an increased generation of ROS by both mesothelial and BEAS-2B cells after exposure to talc (Figures 3A and 3B). This cellular generation of oxidants after exposure to particles was both time-dependent and concentration-dependent, supporting a capacity of cells to produce ROS after exposure to talc. RNA for heme oxygenase and cyclooxygenase, which are potential intracellular sources of oxidative stress, showed conflicting responses, with heme oxygenase significantly increasing in both mesothelial and airway epithelial cells 4 hours after exposure to 100 μg/ml talc, and the concentration of cyclooxygenase not changing (Table E1). Finally, mesothelial and BEAS-2B cells both increased the release of IL-8 after exposure to 100 μg/ml talc (Figure 4A). IL-6 demonstrated a trend toward increasing after exposure to talc, but significant differences were not evident. RNA for IL-8 and IL-6 similarly increased after a 4-hour exposure to talc in both mesothelial cells and BEAS-2B cells (Figure 4B). This increased release of proinflammatory mediators reflects a relevant in vitro biological response in both cell types after talc exposure.

All patients with pleurodesis were male. Their mean age (± SD) was 56 ± 18 years. All but one manifested malignant mesothelioma (the exception underwent surgical intervention for repeated pneumothoraces). Tissue specimens, used as control samples, were collected at the time of pneumonectomy for lung cancer (n = 6) and at autopsy of patients with IPF (n = 6). These individuals manifested no pleurodesis. The specimens obtained during pneumonectomy reflected tissue uninvolved by fibrosis, whereas specimens from patients diagnosed with IPF included fibrotic specimens unrelated to mineral oxide exposure. Control tissue collected during pneumonectomy and autopsy were from men with mean ages (± SD) of 70 ± 15 years and 68 ± 9 years, respectively. Control lungs demonstrated no staining for iron in the pleura or parenchyma (Figure 5A). Significant iron staining occurred in all specimens collected after pleurodesis. Iron was localized to both the parietal (Figure 5B) and visceral (Figure 5C) pleura. Some of the talc was observed to be subpleural (Figure 5C), and particles were also evident in the parenchyma of the lungs (Figure 5D). When particles moved subpleurally and into the parenchyma, they retained their capacity to accumulate the metal (Figure 5D). Some uptake of the ferritin antibody by pleura occurred in the control specimens collected from patients with lung cancer (Figure 5E) and IPF, but this uptake was minimal. Among patients with IPF, uptake for the ferritin body corresponded to areas of inflammation (Figure 5F). After exposure to talc, the staining for ferritin was so intense that the visceral pleura appeared black (Figure 5G). After transport to the lung parenchyma, talc continued to demonstrate an association with an increased expression of ferritin protein (Figure 5H). The expression of this storage protein was focally distributed to the talc, with increased staining immediately adjacent to the particle.

Little staining for DMT1 was evident in the pleura of lungs resected from control patients, and this staining was limited to respiratory epithelia (Figures 6A and 6B). The expression of this major iron importer in the pleura increased enormously after pleurodesis with talc (Figure 6C). Uptake for the iron exporter FPN1 was negligible among lungs resected from control patients (Figures 6D and 6E). Among those patients with talc pleurodesis, the expression of FPN1 was greatly increased at the pleura and in the lung cells immediately adjacent to translocated particles (Figure 6F). Finally, trichrome staining for collagen demonstrated minuscule, subpleural staining in the control specimens taken during pneumonectomy for lung cancer (Figure 7A). In contrast, sheets of collagen were verified by trichrome staining in lung tissue resected after talc pleurodesis (Figure 7B). In the lungs of patients with pleurodesis, the talc appeared at the periphery of the fibrosis (Figure 7B).

The surfaces of silica and silicate particles, including talc, contain some concentration of silanol groups (Si–OH). Si4+ has a high electron affinity, and the Si–O bond consequently has a significant ionic character and an acidic dissociation constant favoring dissociation at physiologic pH (14). The dissociation of silanol groups contributes to a net negative charge on the particle surface, which generates a capacity for the adsorption and exchange of cations (15). The open network of negatively charged silanol groups on a silica and silicate surface presents spaces large enough to accommodate adsorbed metal cations. As a result of its electropositivity, Fe3+ has a high affinity for oxygen-donor ligands (16, 17), and reacts with the silanol group to form a silicato–iron coordination complex (18):

Fe3++m(SiOH)Fe(OSi)m3-m++mH+.

The dose-dependent adsorption of inorganic iron was demonstrated for surface silanol groups on crystalline silicates, with critical stability constants up to 1 × 1017.15 (critical stability constant = 17.15) (19, 20). Our investigation confirmed the capacity of talc to complex iron from an acellular source comparable to other silica and silicate particles (13). The in vitro exposure to talc similarly affected an accumulation of nonheme iron in both mesothelial and airway epithelial cells. Although the source of this accumulated iron was not identified, the talc surface may complex host metal originally associated with ATP, ADP, GTP, citrate, DNA, free amino acids (21), and mitochondrial sources. However, the cell will recognize this loss of requisite iron, and subsequently increases metal import. After integration of the particle's capacity for complexation into the cell's requirement for iron, increased metal uptake and storage would occur. Reflecting these processes, concentrations of ferritin increased after in vitro exposure to talc. As a result of posttranscriptional control acting as a major contributor to expression (2224), the RNA for ferritin, DMT1, and FPN1 did not change in mesothelial and BEAS-2B cells.

Comparable to in vitro accumulation of iron after exposure to talc, significant metal accumulation occurred in vivo after pleurodesis. Perl's Prussian blue staining demonstrated metal in close proximity to retained talc particles in both the mesothelial cells of the pleura and the macrophages and airway epithelial cells of the parenchyma. Because the iron originally associated with the talc approximated normal cell and tissue concentrations, the accumulation of metal after pleurodesis had to originate from host sources of iron. Immunohistochemistry for ferritin showed elevations in expression by mesothelial cells at the pleura and in parenchymal cells after the introduction of talc. The stain for ferritin suggested a very focal response, with cells either directly contacting or immediately adjacent to the particle demonstrating uptake of the antibody to this storage protein. Similarly, the expression of the iron-importer DMT1 and the iron-exporter FPN1 both increased on staining in those patients treated with talc pleurodesis. In control tissue from patients with lung cancer and IPF, no positive staining for iron was evident. The uptake of the antibody to ferritin, DMT1, and FPN1 in these same tissues was minimal, but was evident in mesothelial cells of the visceral pleura, airway epithelial cells, and alveolar macrophages. In addition, ferritin was evident in the chronic inflammatory cells of autopsy samples from patients with IPF. The evidence for disrupted iron homeostasis in IPF was not compelling and this pathway is not proposed as a mechanism of biological effect in all fibrotic injuries.

Elevations in tissue concentrations of ferritin, DMT1, and FPN1 in tissues collected after pleurodesis challenge current understanding, because the controls of expression can be diametrically opposite to each other (22). The expression of these three proteins involves the same posttranscriptional mechanism, using the iron-responsive element (IRE). For ferritin and FPN1, a specific sequence at the 5′-untranslated end of ferritin mRNA (i.e., the IRE) binds a cubane iron–sulfur cluster, referred to as the iron-regulatory protein (IRP), when the IRP exists in the apoprotein form. Elevated concentrations of available iron react with IRP to alter its conformation, decrease affinity of the protein to the mRNA, and displace it from the mRNA, allowing translation to proceed. In contrast, DMT1 mRNA contains an IRE at the 3′-untranslated region that allows increased synthesis with iron depletion. The elevation in expression of all three of these proteins after pleurodesis supports an iron depletion that occurs after the complexation of essential cell metal by the surface of the endocytosed talc particle. This initiates an increase in intracellular iron as the cell elevates concentrations to meet the demands of a new equilibrium imposed by the talc. Subsequently, all three proteins can be elevated, but such expression may involve temporal and regional variability.

The disruption of cell iron homeostasis is frequently associated with oxidative stress (25, 26). In a similar manner, in vitro exposure to talc increased the generation of oxidants, measured as DCF diacetate fluorescence, by mesothelial and airway epithelial cells. The acellular assay for hydroxyl radical production suggests that surface functional groups on the talc complex a source of available iron in the cell, which then redox cycles, generating oxidant. An alternative proposal could involve talc sequestering cell iron and the host response involving superoxide generation as a ferrireductant to resecure the requisite metal. In vitro changes in heme oxygenase RNA after cellular exposures to talc also support oxidative stress.

The biological effect of talc was evaluated using indices of the fibro-inflammatory response. The in vitro cellular response to talc included the increased release of IL-8 and an elevation of RNA for both IL-8 and IL-6, comparable to those for many particles (27). Among patients treated with talc pleurodesis, the deposition of collagen was obvious on trichrome staining, again analogous to numerous particle exposures (6). The indices of both inflammation and fibrosis after exposure to mineral oxide particles were demonstrated to correlate with changes in iron homeostasis. The release of inflammatory mediators can directly correspond to the concentration of metal complexed to a particle surface (28). Regarding the relationship between iron and fibrotic injury, exposure to metal chelators such as bleomycin can increase the activity of prolyl hydroxylase, resulting in a deposition of collagen (29). In vitro and in vivo exposures both confirm that biological effects after talc include those proinflammatory and fibrotic events previously associated with mineral oxide particles.

We conclude that exposure to talc disrupts iron homeostasis in mesothelial and airway epithelial cells, and is associated with both oxidative stress and a biological effect, comparable to those of other particles. The resultant accumulation of iron and alterations in iron-related proteins are evident among patients with pleurodesis. Sclerosis after exposure to talc can be regarded as a model of therapeutic benefit after a massive and focal exposure to a mineral oxide particle.

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Correspondence and requests for reprints should be addressed to Andrew Ghio, Division of Environmental Public Health, Human Studies Facility, United States Environmental Protection Agency, Campus Box 7315, 104 Mason Farm Road, Chapel Hill, NC 27711. E-mail:

Disclaimer: This report was reviewed by the National Health and Environmental Effects Research Laboratory of the United States Environmental Protection Agency and was approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2011-0168OC on November 17, 2011

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