Abstract

Abnormally high incidences of asbestos-related pulmonary disease have been reported in residents of Libby, Montana, because of occupational and environmental exposure to asbestos-contaminated vermiculite. The mechanism by which Libby amphibole (LA) causes pulmonary injury is not known. The purpose of this study is to compare the cellular stress responses induced in primary human airway epithelial cells (HAECs) exposed to a respirable size fraction (≤ 2.5 μm) of Libby amphibole (LA2.5) to a similar size fraction of a reference amphibole sample amosite (AM2.5). HAEC were exposed to 0, 2.64, 13.2, or 26.4 μg/cm2 AM2.5 or LA2.5 or to equivalent doses of unfractionated amosite (AM) or LA for 2 or 24 h. Comparable messenger RNA transcript levels were observed for interleukin-8, cyclooxygenase-2, and heme oxygenase-1 in HAEC following a 24-h exposure to AM or LA. Conversely, exposure to AM2.5 resulted in a 4- to 10-fold greater induction in these proinflammatory mediators compared with LA2.5 after 24 h. Evaluation of the expression of 84 additional genes involved in cellular stress and toxicity responses confirmed a more robust response for AM2.5 compared with LA2.5 on an equal mass basis. Differences in total surface area (TSA) by gas adsorption, total particle number, or oxidant generation by the size-fractionated particles did not account for the observed difference in response. In summary, AM2.5 and LA2.5 are at least as potent in stimulating production of proinflammatory cytokines as unfractionated AM and LA. Interestingly, AM2.5 was more potent at inducing a proinflammatory response than LA2.5. This difference could not be explained by differences in mineral contamination between the two samples, TSA, or oxidant generation by the samples.

The community of Libby, Montana, has been the focus of recent national attention because of the high prevalence of pulmonary disease as a result of occupational and environmental exposure to asbestos-contaminated vermiculite mined in the area prior to 1990 (Horton et al., 2006; McDonald et al., 2003; Sullivan, 2007; Whitehouse, 2004; Whitehouse et al., 2008). Although lung cancer mortality was found to be elevated by 20–30% in Libby mine workers, asbestosis mortality rates in the Libby workers were the most striking with reported increases of 40–80 times the U.S. national average (ATSDR, 2002; Sullivan, 2007). Morphological and compositional analysis of the contaminating asbestos collected from the vermiculite mine identified it as a complex mixture of both regulated and nonregulated amphibole mineral types, primarily winchite, richterite, and tremolite (Meeker et al., 2003). This unique sample composition as well as the associated epidemiological evidence for health effects related to amphibole exposure in the Libby community has prompted a comprehensive toxicological assessment of the Libby amphibole (LA).

Approximately 20% of inhaled elongate minerals are retained in the respiratory tract, primarily in the tracheobronchial and alveolar regions (Asgharian and Yu, 1988). Thus, epithelial cells lining the respiratory tract are one of the first cell types to come into contact with inhaled asbestos particles in vivo. Although the fraction of particles deposited in the alveolar region (∼8–12%) is greater than that deposited in the tracheobronchial region (∼4–7%), the much larger surface area in the alveolar region (500,000 vs. 4800 cm2) equates to a significantly lower effective dose of particles per unit surface area (Asgharian and Yu, 1988). Thus, bronchial epithelial cells experience the highest effective dose of asbestos particles, especially at the junction of the terminal bronchial and alveolar region where asbestos deposition is most concentrated (Brody and Roe, 1983; Brody et al., 1981, 1984).

Epithelial cells, along with alveolar macrophages, are implicated in the onset of pulmonary fibrosis such as asbestosis, through the secretion of proinflammatory cytokines and growth factors such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 as well as reactive oxygen species (ROS) which are produced during both acute and chronic inflammatory events (Mills et al., 1999; Murthy et al., 2009; Nishimura et al., 2007). Other proinflammatory mediators including interleukin (IL)-8 and cyclooxygenase (COX)-2 have been shown to be elevated in airway epithelial cells, induced sputum, or in serum of asbestos-exposed individuals diagnosed with asbestosis (Ilavska et al., 2005; Lappi-Blanco et al., 2006; Setta et al., 2008). Correspondingly, increased expression of these mediators has also been reported in airway epithelial cells in vitro in response to asbestos exposure (Leyva and Roberts, 2010; Rosenthal et al., 1994). Because an acute inflammatory response is one of the earliest events in the pathogenesis of pulmonary fibrosis, measurement of these proinflammatory mediators induced in response to amphibole particles in short-term assays may provide insight into the relative fibroproliferative potential of different amphibole types.

The deposition of both spherical and elongate particles in the lung is dependent on size, with smaller particles having a greater probability of reaching the distal airways. Extensive research on ambient air particulate matter has shown that particles with an aerodynamic diameter (Dae) of ≤ 10 μm (thoracic fraction) are respirable in humans and deposit primarily in the tracheobronchial and alveolar regions (Kim, 2009; Venkataraman, 1999). Similarly, studies investigating the deposition patterns of elongate particles in both rodent and human lung tissues and cast models have shown that aerodynamic diameter and length are the primary determinants of deposition into the lower airways (Brody et al., 1984; Dai and Yu, 1998; Lippmann, 1990; Sturm and Hofmann, 2009). Importantly, a correlation has been shown in rat models of asbestosis between deposition patterns and early fibroproliferative responses in epithelial and interstitial cells especially in the bronchoalveolar regions (Brody et al., 1981; McGavran and Brody, 1989).

The amphibole class of asbestiform minerals has a wide range of particle size distributions, some of which are small enough that they would be predicted to deposit deep in the lung. However, investigations into the relative contribution of a smaller “respirable” size fraction of asbestos particles to the overall toxicity have not been previously addressed. The importance of this data gap to the field of asbestos research was recently emphasized in a document drafted by The National Institute of Occupational Safety and Health (NIOSH) entitled “NIOSH Research Roadmap on Asbestos Fibers and Other Elongate Mineral Particles.” Both the NIOSH document and the Institute of Medicine of the National Academies’ review of the NIOSH document (2009) (Institute of Medicine and the National Research Council of the National Academies, 2009) further discussed a critical role for in vitro strategies in accomplishing these research objectives. Although in vitro toxicity assays have recognizable limitations, they are invaluable in their ability to rapidly screen numerous samples with relatively low cost and are a vital tool for future toxicity testing using comparative approaches.

This study focuses on the relative toxicity assessment of LA as measured by gene expression changes of IL-8, COX-2, heme oxygenase (HO)-1, as well as 84 other genes involved in cellular stress –responsive pathways, with comparison to a well-characterized amphibole standard sample, amosite (AM) by exposing cultured primary human bronchial epithelial cells in vitro. In addition to the unfractionated samples, we further compare the relative toxicity of a smaller size fraction (Dae of ≤ 2.5 μm) of each of the amphibole samples. This investigation is one aspect of the Libby Action Plan (U.S. Environmental Protection Agency, 2007), a large multigroup research effort that includes both in vivo rat instillation and inhalation studies, with which the results of these in vitro experiments will eventually be correlated.

MATERIALS AND METHODS

Amphibole samples.

LA was collected from the Rainy Creek Complex near Libby, Montana, in the year 2000 and processed by U.S. Geological Survey (USGS, Denver, CO). The AM reference sample was obtained from the Research Triangle Institute (Research Triangle Park, NC). Union International Centere le Cancer (UICC) crocidolite (CRO) was also included in select portions of the study as an additional reference sample. A portion of the LA and AM samples was size fractionated by water elutriation using a method adapted from Webber et al. (2008) to isolate particles having an aerodynamic diameter (Dae) of ≤ 2.5 μm from larger particles based on settling velocity, which is a function of the particle density. The Webber methodology takes into account the elongated shape of amphibole particles in the calculation of aerodynamic diameter. Although the unfractionated LA sample is a mixture of different amphibole types including winchite, richterite, and tremolite, preferential enrichment of any of these amphiboles in the elutriated sample is not likely because all amphiboles have very similar densities and aerodynamic properties. Following the elutriation process, the size-fractionated particles were collected on a nylon filter with a 0.22-μm pore size and dried overnight. The size-fractionated amosite and Libby amphibole samples are referred to as AM2.5 and LA2.5, respectively. Unfractionated samples are referred to as AM and LA.

Scanning electron microscopy (SEM) images were acquired for AM, LA, AM2.5, and LA2.5 (Fig. 1). Gold-coated specimens were examined in a scanning electron microscope (JEOL 6400 SEM; JEOL USA, Inc.) equipped with a 30-mm2 energy-dispersive x-ray detector, pulse processor, power supply, and analyzer (Link, 2000; Oxford Analytical Systems, High Wycombe, UK) with interface to an electronic multichannel analyzer (Spectral Engine; 4pi, Inc., Durham, NC) and microcomputer (Macintosh Quadra 650; Apple Computer, Cupertino, CA). An area was selected for analysis, and secondary electron images were obtained for reference.

FIG. 1.

SEM images taken before and after size fractionation by water elutriation to generate samples with an aerodynamic diameter equivalent of ≤ 2.5 μm. (A) Unfractionated AM, (B) size-fractionated AM2.5, (C) unfractionated LA, and (D) size-fractionated LA2.5. Scale bars are 10 μm.

FIG. 1.

SEM images taken before and after size fractionation by water elutriation to generate samples with an aerodynamic diameter equivalent of ≤ 2.5 μm. (A) Unfractionated AM, (B) size-fractionated AM2.5, (C) unfractionated LA, and (D) size-fractionated LA2.5. Scale bars are 10 μm.

Characterization of size-fractionated amphibole samples.

Particle size distributions (length, width, and aspect ratio) were counted for ∼1000 particles for the AM2.5 and LA2.5 samples using SEM (Supplementary tables S1 and S2 [Lowers and Bern, 2009]). All objects were included in the count regardless of dimension, morphology, or mineralogy. Mineral identification was obtained by a combination of energy-dispersive x-ray spectroscopy (EDS) and x-ray diffraction analysis using standard reference samples for comparison. Table 1 summarizes the length, width, and aspect ratio data for the size-fractionated samples as well as the percentage of amphibole and non-amphibole minerals in each sample. Total surface area (TSA) of all samples was measured by krypton gas adsorption using Brunauer-Emmett-Teller (BET) theory (Micromeritics Analytical Services, Norcross, GA). Reported surface areas (Table 2) are an average of two replicate measurements.

TABLE 1

Particle Size Distribution Summary for the Size-Fractionated Amosite (AM2.5) and Libby Amphibole (LA2.5) Samples

Property AM2.5 LA2.5 
Total objects counted (n967 1175 
    Amphiboles (% total) 878 (91) 1082 (92) 
    Non-amphibole minerals (% total) 85 (9) 93 (8) 
Length (μm)a   
    Mean ± SD 6.9 ± 12 1.9 ± 2.1 
    Median 2.9 1.3 
    Range 0.2–104 0.2–24 
Width (μm)a   
    Mean ± SD 0.3 ± 0.2 0.3 ± 0.2 
    Median 0.3 0.3 
    Range 0.02–1.8 0.02–2.6 
Aspect ratioa   
    Mean ± SD 24 ± 37 7.1 ± 8.3 
    Median 11 4.3 
    Range 1.0–360 1.0–129 
Property AM2.5 LA2.5 
Total objects counted (n967 1175 
    Amphiboles (% total) 878 (91) 1082 (92) 
    Non-amphibole minerals (% total) 85 (9) 93 (8) 
Length (μm)a   
    Mean ± SD 6.9 ± 12 1.9 ± 2.1 
    Median 2.9 1.3 
    Range 0.2–104 0.2–24 
Width (μm)a   
    Mean ± SD 0.3 ± 0.2 0.3 ± 0.2 
    Median 0.3 0.3 
    Range 0.02–1.8 0.02–2.6 
Aspect ratioa   
    Mean ± SD 24 ± 37 7.1 ± 8.3 
    Median 11 4.3 
    Range 1.0–360 1.0–129 
a

Reported length, width, and aspect ratios were calculated using amphibole data only and excluded the non-amphibole minerals.

TABLE 2

Total Surface Area Measured by Krypton Gas Adsorption Using BET Theory

Asbestos sample Total surface area (m2/g) ± SEM 
Unfractionated AM 3.1 ± 0.05 
Unfractionated LA 5.3 ± 0.23 
Size-fractionated AM2.5 9.0 ± 0.11 
Size-fractionated LA2.5 12.9 ± 0.42 
Asbestos sample Total surface area (m2/g) ± SEM 
Unfractionated AM 3.1 ± 0.05 
Unfractionated LA 5.3 ± 0.23 
Size-fractionated AM2.5 9.0 ± 0.11 
Size-fractionated LA2.5 12.9 ± 0.42 
Cellular exposure to amphibole particles.

Primary human airway epithelial cells (HAECs) were obtained by brush biopsy of the mainstem bronchus of healthy, nonsmoking adult volunteers (n = 8; 6 male, 2 female; median age = 26 [20–33]) undergoing routine fiber-optic bronchoscopy for the sole purpose of sample collection for scientific study. The cells obtained from this procedure are representative of those cells populating the airways down through the terminal bronchiole region. The use of primary cells as opposed to established cell lines negates any concerns as to whether transformation or immortalization alters the response to these particles and as a result is expected to more accurately relate to in vivo responses for this same cell type. The human subject protocol under which these cells were obtained was reviewed and approved by the Human Subjects Institutional Review Board at the University of North Carolina at Chapel Hill as well as the EPA. HAECs were grown on Corning Costar plastic tissue culture plates (Corning, Inc. Wilkes-Barre, PA) in supplemented bronchial epithelial cell basal medium (BEGM) (Clonetics, San Diego, CA), as described previously (Tal et al., 2006). Briefly, the cells collected on the brush were plated on plastic tissue culture plates and grown and expanded submerged under media until passage 3 at which point they were exposed to the asbestos particles. During the expansion process, only nondifferentiated cells have the capability of dividing and any terminally differentiated cells (mucin-producing cells, ciliated cells, etc.) do not survive. Thus, although the initial cell population obtained by the brush biopsy will be variable between different volunteer subjects, the end result of all cultures is a homogeneous monolayer of a single epithelial cell population, which is consistent between different donor cells. This methodology of cell culture has been used for many years by numerous researchers involved with in vitro toxicology studies of particulates (Becker et al., 2005; Frampton et al., 1999; Graff et al., 2007; Molinelli et al., 2002; Samet et al., 1996; Tal et al., 2006; Wu et al., 2001).

Amphibole samples were prepared fresh as 2 mg/ml stock solutions in sterile water. The size-fractionated samples were sonicated by cup horn sonication before cell exposure to break up the tightly packed particles that resulted from the filtration process. The unfractionated samples were vigorously vortexed to disperse the particles but were not sonicated to avoid inadvertent breakage of the longer fibers. HAECs were exposed as a confluent monolayer to 0, 2.64, 13.2, or 26.4 μg/cm2 of CRO, AM, AM2.5, LA, or LA2.5 for 2 or 24 h. Cell supernatants were removed and retained for protein analysis. Cells were washed two times with 1× PBS (Gibco-BRL, Gaithersburg, MD) and the cells lysed by the addition of guanidine isothiocyanate and dislodged from the plate with a cell scraper. The cell lysates were subsequently sheared three to five times with a 1-cc syringe and 21-guage needle, and the lysates were stored at −80°C until processed for RNA isolation. Dose-response studies with the unfractionated samples were carried out with three biological replicates in which cells from three different volunteers were used (n = 3), whereas the dose-response studies with the size-fractionated samples were carried out with four biological replicates (n = 4). Within each biological replicate, each exposure condition was repeated with at least two technical replicates.

Cytotoxicity studies.

Supernatants collected from treatment and control wells after 2- and 24-h exposures to the maximum concentration of 26.4 μg/cm2 AM, AM2.5, LA, or LA2.5 were analyzed for cellular leakage of lactate dehydrogenase (LDH) protein using a Cytotox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI) as per the manufacturer's instructions. LDH protein concentrations were expressed as a percentage of LDH detected in the cell culture medium relative to the total LDH present in the cell lysates and the supernatant combined. Supernatant LDH protein levels were ≤ 10% of total cellular LDH following a 24-h exposure of the airway epithelial cells to the maximum mass dose of 26.4 μg/cm2 of any amphibole sample (Supplementary fig. S1). Unexposed cells had 3–5% LDH present in the supernatant.

Secreted proteins are known to adhere to particulates and interfere with protein measurements; therefore, cytotoxicity was also assessed by a complementary assay measuring intracellular calcein fluorescence emitted by live cells. HAECs were exposed to 26.4 μg/cm2 AM2.5 or LA2.5 with and without saponin (0.1%) or saponin alone for 2 or 24 h, and cytotoxicity was measured using a Live/Dead viability/cytotoxicity kit as per manufacturer's instructions (Invitrogen, Eugene, OR). Saponin was used as a positive control to induce cell death in the presence of the amphibole particles. Intracellular levels of calcein fluorescence were measured using a plate reader (BMG Labtech, Durham, NC). The maximum dose of AM2.5 and LA2.5 resulted in 95 and 99% viability, respectively, relative to the untreated control cells (100%) after 2 h and 86 and 94% viability, respectively, after 24 h (Supplementary fig. S1). These values are comparable to other reports of bronchial epithelial cells exposed to CRO standard reference samples (Lang et al., 2001). One-way ANOVA analysis with Dunnett's posttest did not detect a statistically significant difference between exposed cell viability and the untreated control cells (p > 0.05). Taken together, these results confirm that the mass doses of amphibole particles used in this study did not significantly alter the viability of the airway epithelial cells following exposures lasting up to 24 h.

Quantitative RT-PCR.

Dose-dependent relative gene expression changes of the inflammatory markers IL-8, COX-2, and HO-1 in HAECs was quantified using reverse transcriptase (RT)-PCR following a 2- or 24-h exposure to unfractionated AM, LA, or CRO or the size-fractionated AM2.5 or LA2.5. Additionally, the peptide growth factor TGF-β1 messenger RNA (mRNA) was quantified for the AM2.5- and LA2.5-exposed cells after 2 and 24 h. Total RNA was isolated using an RNeasy Mini Kit according to manufacturer's instructions (Qiagen, Valencia, CA), and 100 ng was reverse transcribed to generate complementary DNA (cDNA) using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative fluorogenic amplification of cDNA was performed using primer/probe sets of interest, TaqMan Universal PCR Master Mix, and the ABI Prism 7500 Sequence Detection System (Applied Biosystems). The relative abundance of the mRNA levels was determined from standard curves generated from a serially diluted standard pool of cDNA prepared from cultured airway epithelial cells. mRNA levels were quantified for each experimental replicate and the resultant data combined to give the average fold changes reported in Figure 2.

FIG. 2.

Relative mRNA levels encoding the proinflammatory markers IL-8 (A, B) and COX-2 (C, D) and oxidative stress–responsive marker HO-1 (E, F) following exposure of HAECs to various concentrations of unfractionated AM and LA (A, C, and E) or size-fractionated AM2.5 and LA2.5 (B, D, and F) for 24 h. White bars = 2.64 μg/cm2; gray bars = 13.2 μg/cm2; black bars = 26.4 μg/cm2. mRNA transcript levels were measured by RT-PCR and data expressed as a fold-change difference relative to the no-treatment control. “*” Indicates significant difference between treatment and no-treatment control; “#” indicates significant difference between amosite and Libby amphibole samples at equal concentration; “†” indicates significant difference between AM and CRO at equal concentration. Difference is deemed significant when p < 0.05.

FIG. 2.

Relative mRNA levels encoding the proinflammatory markers IL-8 (A, B) and COX-2 (C, D) and oxidative stress–responsive marker HO-1 (E, F) following exposure of HAECs to various concentrations of unfractionated AM and LA (A, C, and E) or size-fractionated AM2.5 and LA2.5 (B, D, and F) for 24 h. White bars = 2.64 μg/cm2; gray bars = 13.2 μg/cm2; black bars = 26.4 μg/cm2. mRNA transcript levels were measured by RT-PCR and data expressed as a fold-change difference relative to the no-treatment control. “*” Indicates significant difference between treatment and no-treatment control; “#” indicates significant difference between amosite and Libby amphibole samples at equal concentration; “†” indicates significant difference between AM and CRO at equal concentration. Difference is deemed significant when p < 0.05.

Measurement of secreted IL-8 protein.

IL-8 protein analysis was carried out using an ELISA on cell supernatants following a 24-h exposure to 2.64, 13.2, or 26.4 μg/cm2 AM2.5 or LA2.5 using a Quantikine Human CXCL8/IL-8 Immunoassay Kit (R&D Systems, Inc., Minneapolis, MN). Data were acquired for three biological replicates in which cells from a different volunteer were used for each replicate (n = 3). Each exposure condition was repeated in duplicate within each biological replicate. Data from each experimental replicate were acquired individually and the results averaged and reported relative to the untreated control.

Measurement of ionizable and chelatable surface iron concentrations.

Ionizable iron concentrations associated with each of the amphibole samples were quantified (Borggaard, 1988; Roth et al., 1969). One milligram of each of the four samples was suspended in 1.0 ml of 3.0 N HCl and agitated in a water bath at 70°C for 60 min. After centrifugation at 1500 × g for 10 min, iron in the supernatant was assayed using inductively coupled plasma optical emission spectroscopy (ICP-OES; Model Optima 4300D; Perkin Elmer, Norwalk, CT) operated at a wavelength of 238.204 nm. Ionizable iron was measured in triplicate and repeated once.

Concentrations of surface-chelatable iron were also measured using a second assay employing citrate-bicarbonate-dithionite (CBD) methodology. In this assay, surface ferrite ions are reduced to ferrous ions which are then able to be chelated by the citrate ligand and subsequently assayed in the reaction supernatant. One milligram of each of the four amphibole samples was exposed to 0.3M sodium citrate, 1.0M sodium bicarbonate, and 10 mg sodium dithionite. This suspension was agitated in a water bath at 70°C for 30 min and centrifuged at 1500 × g for 10 min, and the supernatant was assayed for iron using ICP-OES. Surface iron was quantified in triplicate and the experiment repeated once.

The concentration of water-soluble iron associated with the amphibole particles was quantified. One milligram of each amphibole sample was suspended in 1.0 ml distilled water and agitated for 60 min (n = 5/sample). After centrifugation at 1500 × g for 10 min, iron in the supernatant was assayed using ICP-OES operated at a wavelength of 238.204 nm.

Oxidant generation measured by thiobarbituric acid–reactive products of deoxyribose.

Oxidant generation by each of the four amphibole samples was measured employing thiobarbituric acid (TBA)–reactive products of deoxyribose as previously described (Halliwell and Gutteridge, 1981). Briefly, 2-deoxy-D-ribose reacts with an unspecified iron-catalyzed oxidant, or oxidants, to form a mixture of malondialdehyde adduct species. After heating with TBA at a low pH, this compound, or compounds, generates a pink chromogen indistinguishable from a TBA-malondialdehyde adduct which can be used as an assay for hydroxyl radical production by measuring its absorbance at 532 nm. Incubations were repeated with inclusion of either the iron chelator deferoxamine (DFO) or the radical scavenger dimethylthiourea (DMTU) (both at 1.0mM). Measurements were done in triplicate and repeated twice.

Quantitative RT-PCR arrays.

Relative gene expression changes of 84 genes involved in cellular stress and toxicity was quantified using RT-PCR in a 96-well array format following a 24-h exposure of HAECs to 13.2 μg/cm2 AM, LA, AM2.5, or LA2.5 or to 26.4 μg/cm2 LA2.5 only. The Human Stress and Toxicity Pathway Finder RT2 Profiler PCR array (Cat. no. PAHS-003 A; SABiosciences Corporation, Frederick, MD) was used in this study. cDNA equivalent to 500 ng of total RNA was used for each array. The arrays were run on an ABI prism 7500 Sequence Detection System (Applied Biosystems) using the recommended cycling program. Data were normalized to the average Ct of five housekeeping genes (B2M, HPRT1, RPL13A, GAPDH, and ACTB) and expressed with respect to the untreated control. RT-PCR arrays were evaluated for a single technical replicate from at least three independent experiments using cells obtained from different volunteers (n = 3). The resultant Ct values were combined to calculate the average fold regulation values depicted in Figure 4 (Supplementary table S3).

FIG. 3.

Determination of surface iron concentrations and corresponding oxidant generation by each amphibole mineral type. (A) Ionizable iron concentrations measured by ICP-OES following treatment of the samples with 3 N HCl. (B) Surface-chelatable iron concentrations measured by ICP-OES following treatment of the samples with CBD. (C) Spectrophotometric assay measuring the TBA-reactive products of deoxyribose as a measure of hydroxyl radical production. Black bars = unfractionated samples; gray bars = fractionated samples. “*” Indicates significant difference between unfractionated and size-fractionated samples within the same amphibole type; “#” indicates significant difference between amosite and Libby amphibole when comparing the same size fraction.

FIG. 3.

Determination of surface iron concentrations and corresponding oxidant generation by each amphibole mineral type. (A) Ionizable iron concentrations measured by ICP-OES following treatment of the samples with 3 N HCl. (B) Surface-chelatable iron concentrations measured by ICP-OES following treatment of the samples with CBD. (C) Spectrophotometric assay measuring the TBA-reactive products of deoxyribose as a measure of hydroxyl radical production. Black bars = unfractionated samples; gray bars = fractionated samples. “*” Indicates significant difference between unfractionated and size-fractionated samples within the same amphibole type; “#” indicates significant difference between amosite and Libby amphibole when comparing the same size fraction.

FIG. 4.

Gene expression heat maps for each of the seven stress-responsive pathways present on the Human Stress and Toxicity Pathway Finder RT2 PCR Array (SABiosciences). mRNA transcript levels for each of the 84 genes was quantified following treatment of HAECs to 13.2 μg/cm2 size-fractionated AM2.5 (column 1) or LA2.5 (column 2) or unfractionated AM (column 3) or LA (column 4) for 24 h. Fold increase or decrease was determined with respect to the no-treatment control.

FIG. 4.

Gene expression heat maps for each of the seven stress-responsive pathways present on the Human Stress and Toxicity Pathway Finder RT2 PCR Array (SABiosciences). mRNA transcript levels for each of the 84 genes was quantified following treatment of HAECs to 13.2 μg/cm2 size-fractionated AM2.5 (column 1) or LA2.5 (column 2) or unfractionated AM (column 3) or LA (column 4) for 24 h. Fold increase or decrease was determined with respect to the no-treatment control.

Confocal microscopy.

Primary HAECs were cultured on 22-mm diameter glass coverslips placed in 6-well Corning Costar plastic tissue culture plates (Corning, Inc.) in BEGM (Clonetics). Cells were exposed at confluence to 26.4 μg/cm2 of AM2.5 or LA2.5 for 4 or 24 h. After incubation, cells were rinsed with PBS buffer and fixed with 4% paraformaldehyde. Coverslips were mounted onto microscope slides using ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (Agilent Technologies, Santa Clara, CA). Images were taken using a Nikon Eclipse C1Si confocal microscope (Nikon Instruments Incorporation, Melville, NY) equipped with a dark-field condenser (TI DF) and either a ×100.0/1.30 (Fig. 5A) or a ×60.0/1.25 (Figs. 5B and 5C) oil objective. Images were acquired in the plane in which the DAPI stain of the nucleus was in focus, indicating that any fibers that were also in focus in this plane are presumed to be located within the cell.

FIG. 5.

Confocal images visualizing amphibole particle uptake by primary airway epithelial cells. HAECs were exposed to 26.4 μg/cm2 of the size-fractionated LA2.5 for (A) 4 h or (B, C) 24 h. Scale bars are (A) 20 μm, (B) 10 μm, and (C) 25 μm. DAPI staining of the nucleus is shown in blue in the images and the amphibole particles appear as white.

FIG. 5.

Confocal images visualizing amphibole particle uptake by primary airway epithelial cells. HAECs were exposed to 26.4 μg/cm2 of the size-fractionated LA2.5 for (A) 4 h or (B, C) 24 h. Scale bars are (A) 20 μm, (B) 10 μm, and (C) 25 μm. DAPI staining of the nucleus is shown in blue in the images and the amphibole particles appear as white.

Statistical analysis.

Data are presented as mean ± SEM unless otherwise indicated. Quantitative RT-PCR data for IL-8, COX-2, and HO-1 were evaluated by both one-way ANOVA using the Dunnett post hoc procedure for comparison between treatment and control cells as well as two-way ANOVA using the Bonferroni posttest for comparison between the different amphibole types (AM, LA, and CRO) at the same mass dose. RT-PCR array data were analyzed using the RT2 Profiler PCR Array Data Analysis software (SABiosciences). Surface iron, water-soluble iron, and oxidant generation data were evaluated by paired t-tests between unfractionated and size-fractionated samples. Significance was assumed at p < 0.05.

RESULTS

Size-Fractionation and Characterization of Amphibole Samples

Elutriated particles comprised 12% by weight of unfractionated AM and 17% by weight of the unfractionated LA. Figure 1 compares SEM images obtained for the unfractionated AM (Fig. 1A) or LA (Fig. 1C) with the corresponding size-fractionated samples (AM2.5 or LA2.5, respectively) (Figs. 1B and 1D). These images show a visual difference in particle size distribution between the unfractionated and size-fractionated samples with the majority of the longer and thicker particles having been removed by the elutriation process, thus confirming successful isolation of a smaller size fraction of these two amphibole samples. Table 1 summarizes the length, width, and aspect ratio values for each of the size-fractionated amphibole samples (AM2.5 and LA2.5) as determined by SEM analysis of approximately 1000 objects (Supplementary tables S1 and S2). All particles were included in the SEM count without any length, aspect ratio, or mineralogy restrictions; however, the values reported in Table 1 for length, width, and aspect ratio were calculated using only the data acquired from those particles classified as an amphibole mineral and excluded any contribution from the non-amphibole minerals present in the sample. Both samples contained < 10% of non-amphibole minerals including, but not limited to, biotite/phlogopite (3.0%), quartz (2.0%), and Fe-silicate (1.6%) in the AM2.5 sample and K-feldspar (3.0%), quartz (0.9%) and vermiculite (0.9%) in the LA2.5 sample. Examination of the amphibole size distribution data in Table 1 shows comparable width values between the two size-fractionated samples, whereas the AM2.5 sample consists on average of longer particles compared with the LA2.5 sample. As a result, the AM2.5 sample has higher mean and median aspect ratios compared with the LA2.5 sample. These dimensional differences are visually apparent in the corresponding SEM images presented in Figure 1.

Using the data acquired in the SEM analysis of the size-fractionated samples, the total number of amphibole particles per milligram of sample was determined to be relatively consistent between the two amphibole samples with calculated values of 7.1 × 108 and 7.9 × 108 particles/mg for AM2.5 and LA2.5, respectively. Reported in Table 2 are the TSA measurements for each of the unfractionated and size-fractionated samples measured by krypton gas adsorption and BET analysis. The TSA of the unfractionated samples was found to be lower than the corresponding size-fractionated samples and both the unfractionated and size-fractionated Libby amphibole had higher TSA compared with the corresponding amosite samples.

Size-Fractionated AM2.5 Induces a Greater Proinflammatory Response than LA2.5

IL-8, whose role in vivo is to recruit neutrophils to the site of injury, is frequently reported to be upregulated in airway epithelial cells in response to insult by a multitude of particles both in vivo and in vitro and is thus an appropriate reporter of an acute proinflammatory response in in vitro assays (Becker et al., 2005; Donaldson et al., 2008; Duvall et al., 2008; Park et al., 2008; Rouse et al., 2008; Tal et al., 2010). COX-2 is another important proinflammatory mediator that is induced in response to cell stress and injury by cellular pathways distinct from IL-8 (Becker et al., 2005; Mitchell et al., 1994). Lastly, HO-1 is a stress-inducible enzyme that is involved in cytoprotection as well as apoptosis and inflammation (Gozzelino et al., 2010). The relative expression of these three indicators was measured in this study as a means of assessing the magnitude of the acute proinflammatory response in HAECs exposed to amphibole particles.

HAECs were exposed on an equivalent mass basis to 0, 2.64, 13.2, or 26.4 μg/cm2 of unfractionated AM or LA or size-fractionated AM2.5 or LA2.5 for 2 or 24 h. Additionally, cells were exposed to the same concentrations of CRO for 24 h for additional comparison to another well-known standard reference sample. Relative expression of mRNAs coding for IL-8, COX-2, and HO-1 were quantified by RT-PCR and the acquired data plotted as fold change relative to the untreated control.

Following a 2-h exposure, IL-8 mRNA transcript levels were minimally elevated (fold change of 2.5) in response to the maximum concentration (26.4 μg/cm2) of the unfractionated LA; however, no significant change in IL-8 expression was detected in the AM-treated cells (Supplementary fig. S2). COX-2 and HO-1 mRNA levels were also not significantly elevated following a 2-h exposure to either of the unfractionated AM or LA samples (Supplementary fig. S2). Similarly, exposure of HAECs to the size-fractionated AM2.5 or LA2.5 for 2 h induced minimal increases in mRNA expression of IL-8, COX-2, or HO-1 (Supplementary fig. S2).

Conversely, a clear dose-response was observed for all genes under investigation after a 24-h treatment of HAECs to each of the amphibole samples (Fig. 2). Unfractionated AM- and LA-treated cells responded comparably with similar increases in the relative expression of the proinflammatory and oxidative stress–responsive genes at equal concentration. Specifically, maximum fold-change values of 50 ± 7.5 and 46 ± 8.3 for IL-8 mRNA (Fig. 2A), 5.4 ± 0.5 and 9.0 ± 1.7 for COX-2 mRNA (Fig. 2C), and 2.9 ± 0.2 and 2.5 ± 0.2 for HO-1 mRNA (Fig. 2E) were detected in response to 26.4 μg/cm2 of unfractionated AM and LA, respectively. These responses were also comparable to the CRO-exposed cells at the same concentration. Although significant differences were detected in COX-2 mRNA expression for the highest concentration between AM- and LA-treated cells, as well as between AM- and CRO-treated cells, these differences were relatively small compared with those observed between the size-fractionated samples.

Exposure of HAECs to the smaller size fractions resulted in significant differences in the magnitude of the proinflammatory response measured by the gene expression changes of IL-8 and COX-2. The size-fractionated AM2.5 caused a fourfold greater induction in IL-8 mRNA expression and a 10-fold greater induction in COX-2 mRNA expression compared with those cells treated with an equal concentration of size-fractionated LA2.5. Specifically, maximum fold-change values of 120 ± 25 and 37 ± 7.8 for IL-8 mRNA (Fig. 2B) and 16 ± 2.8 and 1.6 ± 0.3 for COX-2 mRNA (Fig. 2D) were detected following exposure to 26.4 μg/cm2 of the size-fractionated AM2.5 and LA2.5, respectively. Interestingly, HO-1 gene expression was equally upregulated by the two amphiboles with maximum fold-change values of 4.5 ± 0.3 and 5.1 ± 0.6 for the same concentration of AM2.5 and LA2.5, respectively.

Despite the known possibility of particle interference in protein measurements, IL-8 protein levels were quantified in the cell supernatants using an ELISA assay (Supplementary fig. S3). As anticipated, exposure of the cells to the two highest concentrations (13.2 and 26.4 μg/cm2) of both AM2.5 and LA2.5 resulted in statistically significant increases in IL-8 compared with control values (8.8 ± 1.7 and 10 ± 1.9 for AM2.5 and 4.0 ± 0.7 and 5.7 ± 1.1 for LA2.5), but these increases were much smaller than would have been expected based on changes in IL-8 mRNA. Because of the likelihood of interference with ELISA assays, only mRNA changes are reported here.

Surface Iron Measurements and Related Oxidant-Generating Potential of Amphibole Particles

The pathogenicity of asbestos particles is thought by many to be due, at least in part, to the ability of the minerals to generate ROS via Fenton chemistry at the surface of the silicate mineral (Kamp et al., 1992; Kennedy et al., 1989). Furthermore, the degree of oxidant production has been shown to be proportional to the concentration of surface-complexed iron. To determine if the difference in inflammatory response between AM2.5 and LA2.5 could be explained by surface-complexed iron, both ionizable and surface-chelatable iron concentrations were measured in both the unfractionated and size-fractionated Libby amphibole and amosite samples. Figure 3A shows the concentration of ionizable iron measured by ICP-OES following treatment of each amphibole sample with 3 N HCl. This assay ensures complete ionization of surface-complexed iron. Size-fractionated AM2.5 was found to have the highest concentration of ionizable iron, which was significantly greater than the corresponding unfractionated AM sample. In contrast, the Libby amphibole samples contained much lower amounts of ionizable iron, and there was no significant difference in the quantity of ionizable iron between the unfractionated and fractionated samples. A similar trend was observed in the complementary assay measuring surface-chelatable iron by ICP-OES following treatment of each amphibole sample with CBD (Fig. 3B), although in this case the fractionated LA2.5 sample also had significantly higher iron levels than the unfractionated sample.

To ensure that the chemical composition of the size-fractionated amphibole samples was not altered significantly as a result of the water elutriation process, water-soluble iron was quantified by ICP-OES for both the unfractionated and fractionated samples. Unfractionated AM and LA were found to have soluble iron concentrations of 0.05 ± 0.003 ppm and 0.04 ± 0.006 ppm, respectively, whereas the size-fractionated AM2.5 and LA2.5 samples were found to have concentrations equaling 0.04 ± 0.003 ppm and 0.03 ± 0.008 ppm, respectively. Although the size-fractionated samples had slightly less water-soluble iron than their unfractionated counterparts, the difference was not statistically significant (p > 0.05). From this, it was concluded that the water elutriation process did not significantly alter the surface chemistry of the amphibole particles.

Oxidant generation by both size fractions of amosite and Libby amphibole was assessed using a common spectrophotometric assay measuring the production of TBA–reactive products of deoxyribose. The absorbance at 532 nm is related to the amount of hydroxyl radical anion produced in the presence of the amphibole minerals. The results of this assay are presented in Figure 3C, which shows a similar trend between oxidant generation and relative concentrations of surface-complexed iron (Figs. 3A and 3B). Both amosite samples (AM and AM2.5) were found to generate a greater amount of hydroxyl radicals compared with the two Libby amphibole samples (LA and LA2.5). Both of the size-fractionated samples (AM2.5 and LA2.5) had small but significant increases in the amount of ROS produced compared with the respective unfractionated samples. Pretreatment of the samples with an iron chelator, DFO, or a hydroxyl radical scavenger, DMTU, completely inhibited the production of ROS by all amphibole samples (Supplementary fig. S4).

These data might suggest that the greater inflammatory response induced by AM2.5 compared with LA2.5 as measured by gene expression changes might be explained by surface iron or production of ROS. However, it is difficult to reconcile this suggestion with the finding that the unfractionated AM also had significantly more surface iron and produced more ROS than the unfractionated LA, even though these two samples evoked an equivalent inflammatory response in HAECs.

Gene Expression Profiling Using RT-PCR Arrays

To better understand mechanistic differences that may explain the differential response of HAECs to size-fractionated LA2.5 and AM2.5, we expanded our gene expression analysis to include 84 genes known to be involved in specific cellular stress-responsive pathways using the Human Stress and Toxicity Pathway Finder RT2 Profiler PCR array. RT-PCR array technology provides a rapid and simplified approach to assess the relative expression of multiple genes in a more quantitative manner than microarray, with the added advantage of analyzing only a specific subset of genes implicated in cellular pathways of interest. Pathway-focused gene expression heat maps shown in Figure 4 were generated for each of the seven stress-responsive pathways present on the array following a 24-h exposure to 13.2 μg/cm2 of either AM2.5, LA2.5, AM, or LA.

Examination of the heat maps clearly shows a robust proinflammatory gene expression response induced by all four amphibole samples. The other pathways also demonstrated gene expression changes though not to the degree of the inflammatory pathway. Changes in mRNA transcript levels of most genes in these other pathways were altered less than fivefold. Similar to the earlier findings for IL-8 and COX-2, the expanded set of proinflammatory mediators also demonstrated a notable difference in the magnitude of response between the size-fractionated AM2.5 and LA2.5. The greater induction of mRNA transcripts coding for inflammatory genes in response to AM2.5 compared with LA2.5 is exemplified by the top two upregulated genes in the inflammation pathway with fold-change values for TNF mRNA of 23 ± 11 and 12 ± 6.3 for AM2.5 and LA2.5, respectively, and 54 ± 35 versus 6.0 ± 3.7 for CSF2 (granulocyte macrophage colony-stimulating factor [GM-CSF]). Similarly, mRNA transcript levels were markedly elevated for IL-6, IL-1a, IL-1b, and IL-18 in response to AM2.5 compared with LA2.5. In particular, IL-6 mRNA levels were increased 13-fold in response to AM2.5, whereas LA2.5 did not alter the expression of this cytokine at the equivalent concentration. Although mRNA levels for several genes in the inflammatory pathway were increased in response to the size-fractionated amphibole samples, others in this pathway were found to be unaffected, whereas a selected few, including NOS2, were decreased. The AM2.5-treated cells exhibited greater perturbation in these downregulated genes compared with LA2.5-treated cells, confirming an overall more robust response in these cells in response to AM2.5 compared with LA2.5.

Comparison of the expression profiles between AM2.5 and LA2.5 across all pathways on the array revealed differences in the expression pattern of several genes in that mRNA levels were found to be increased with respect to the untreated control in response to one amphibole type but decreased or unaltered in response to the other amphibole type. Included in this list were IL-6 and DNAJB4, whose respective mRNAs were increased compared with the control in response to AM2.5 but were either unaffected or decreased in response to LA2.5. Conversely, mRNAs relating to CXCL10, EPHX2, and CRYAB were decreased compared with control in response to AM2.5 but were unaffected or increased in response to LA2.5. These gene expression differences might be indicative of activation of different cellular pathways in response to these two size-fractionated samples.

In contrast, the gene expression profiles for the unfractionated AM and LA samples were highly complementary with respect to the magnitude of response. In particular, expression of the proinflammatory cytokines demonstrated large similarities with increased transcript levels detected for the genes TNF, CSF2, IL-6, IL-1a, IL-1b, and CXCL10. For comparison to the expression levels reported earlier for AM2.5 and LA2.5, the TNF mRNA levels were increased 24 ± 12 and 21 ± 11-fold in response to the unfractionated AM and LA, respectively, and CSF2 was upregulated 30 ± 8.0 and 17 ± 4.6-fold, respectively. Additionally, IL-6 mRNA transcript levels were increased 4.8 ± 1.8 and 2.9 ± 0.8 for AM and LA, respectively.

Clearly, altered expression of proinflammatory genes was the most robust in response to either the size-fractionated or unfractionated samples. Interestingly, exposure to the amphibole samples resulted in a reduction of mRNA levels compared with the control for many of the genes in these seven pathways, rather than increased mRNA expression. This pattern is especially apparent for the oxidative or metabolic stress pathway and the heat shock pathway genes. Once again, the difference in expression of these genes between size-fractionated samples was much different than that found between the unfractionated samples.

Particle Internalization by Epithelial Cells

Epithelial cells are known to take up particles similar in size to the fractionated samples used in this study. To evaluate particle internalization by the airway epithelial cells, HAECs were exposed as a confluent monolayer to 26.4 μg/cm2 of the size-fractionated AM2.5 or LA2.5, and the cells were imaged using confocal microscopy after 4 and 24 h. Presented in Figure 5 are the resultant images for the LA2.5-exposed epithelial cells in which the nucleus is visualized in blue (DAPI) and the amphibole particles appear as white. Following a 4-h exposure (Fig. 5A), the amphibole particles were found localized primarily on the periphery of the cell with some particles visualized in the plane of the nucleus implying internalization. Interestingly, after 24 h (Figs. 5B and 5C), the particles were nearly all taken up by the epithelial cells and were found to primarily cluster around the nucleus. Exposure of the HAECs to the size-fractionated AM2.5 resulted in a similar temporal pattern of particle uptake with the internalized particles also localizing around the nucleus after 24 h (data not shown).

DISCUSSION

In this study, we investigated the relative toxicity of Libby amphibole collected from the contaminated vermiculite mine located in the Rainy Creek Complex near Libby, Montana, with a well-characterized amphibole sample of amosite by assessing gene expression changes induced in an in vitro cell culture system. Airway epithelial cells were exposed to both unfractionated AM or LA as well as size-fractionated AM2.5 or LA2.5 that had been water elutriated to isolate particles with aerodynamic diameter of ≤ 2.5 μm. The 2.5-μm fraction was included in this investigation to assess the relative contribution of a smaller, respirable size fraction to the overall toxicity of these amphibole particles.

Induction of an acute proinflammatory response following a 24-h exposure was observed for all size fractions as determined by gene expression changes of the proinflammatory and stress-responsive genes IL-8, COX-2, and HO-1. Comparable mRNA transcript levels were detected for IL-8 and COX-2 in response to the unfractionated AM, LA, and CRO. LA2.5 stimulated roughly the same level of mRNAs as the unfractionated samples, but AM2.5 stimulated a 4- to 10-fold greater induction of these two genes than LA2.5. These conclusions were drawn from cellular exposure experiments in which cells from three to four different human volunteers were utilized. Although an n of 3 or 4 may be considered a relatively low number, it was sufficient for detection of statistically significant differences in the assays presented in the current study. Although donor variability is expected when using primary cells, the reproducibility observed in these experiments is primarily because of the relative homogeneity of the cell monolayers generated by growing the cells under submerged culture conditions and the preferential selection of the nondifferentiated cells.

Examination of the physicochemical properties of the size-fractionated samples was carried out to explore metrics that may account for the observed difference in gene expression between AM2.5 and LA2.5. LA has been previously reported to contain significant amounts of non-amphibole minerals including calcite, K-feldspar, talc, vermiculite, and others (Meeker et al., 2003). Although the unfractionated sample characterized in this reference was not the exact sample used in the present study, it highlighted the necessity of assessing the relative proportion of amphibole and non-amphibole minerals in the Libby sample, especially when making comparisons to a standard reference sample such as AM. To address this, the mineralogy of 1000 particles was analyzed by SEM/EDS characterization of the size-fractionated AM2.5 and LA2.5 samples. Less than 10% of the particles in the size-fractionated LA2.5 sample were found to be non-amphibole following the water elutriation process (Table 1). Somewhat surprisingly, the AM2.5 sample also consisted of approximately 10% of non-amphibole minerals, indicating that any observed toxicological difference between these two samples was not likely attributed to differences in the relative percentage of amphibole particles. However, one cannot exclude the possibility that chemical differences in the non-amphibole minerals of each sample could be responsible for the differential response, but the fact that these minerals make up only a small percentage of the sample makes this possibility unlikely.

Although mass is the most common dose metric used in comparative studies, alternative metrics including particle number per unit mass and surface area also require consideration in the interpretation of comparative toxicity data of elongate mineral particles. Fiber number, where fiber is defined as > 3:1 aspect ratio and > 5 μm length, has been suggested by many to be a critical metric when assessing the relative carcinogenicity of asbestos minerals (Stanton et al., 1981); however, the validity of this correlation has more recently been brought into question (Dodson et al., 2003). Furthermore, no correlation has been established that relates a proinflammatory response in epithelial cells to specific particle dimensions. Because amphibole particles of all dimensions have reactive surfaces that may influence and/or contribute to the observed proinflammatory response, the total number of amphibole particles per milligram was alternatively assessed in the current study for each of the size-fractionated samples. Total particle number per milligram was found to be nearly equal between the two size-fractionated amphibole samples indicating that the difference in proinflammatory response was not driven by a difference in amphibole particle dose.

Both the unfractionated and size-fractionated samples were further analyzed for TSA by gas adsorption and subsequent BET analysis (Table 2). The unfractionated samples had similar TSA, which was consistent with the observed proinflammatory gene expression data in which equal masses of these two samples induced similar responses in HAECs. Conversely, LA2.5 had a marginally higher TSA compared with AM2.5, which was not consistent with the gene expression data of IL-8 and COX-2 that showed a 4- to 10-fold greater induction of these proinflammatory mediators in AM2.5-exposed HAECs compared with LA2.5 when dosed on an equal mass basis. Thus, interpretation of the data with respect to either particle number per milligram or TSA does not explain the difference in inflammatory response observed between these two amphibole samples. It should be noted, however, that the non-amphibole minerals, which represent < 10% of the particles will also contribute to the TSA measurement by the gas adsorption methodology. Because the non-amphibole mineral types were found to differ between the two size-fractionated samples, we cannot exclude the possibility that these minor contaminants might have a different surface area per unit mass than the amphibole particles and thus skew the surface area as measured by gas adsorption.

It is widely accepted that amphibole particles are capable of generating ROS via Fenton reactions catalyzed primarily by surface-complexed iron (Ghio et al., 1992a, 1992b). The production of ROS by silicate minerals is one of the mechanisms by which asbestos minerals are proposed to induce toxicity. Surface reactivity was directly quantified for each of the amphibole samples by assessing both the concentration of surface-complexed iron as well as the relative abundance of oxidants produced in an aqueous acellular environment. As anticipated, the relative concentration of surface-complexed iron, which was determined by two complementary assays measuring for ionizable and surface-chelatable iron concentrations, was proportional to the amount of hydroxyl radical anions produced by the amphibole samples (Fig. 3). Although the relative amount of hydroxyl radicals generated by AM2.5 versus LA2.5 correlated with the observed difference in proinflammatory response, this was not true for the unfractionated AM and LA where the large differential in oxidant production was not observed in the proinflammatory response. Furthermore, HO-1 mRNA transcript levels were uniformly upregulated by all size fractions of AM and LA despite the differences in oxidant-generating potential between the amphibole types. Blake et al. (2007) reported similar results with murine alveolar macrophages in which unfractionated LA induced a greater production of intracellular ROS compared with CRO at an equal mass dose despite the fact that CRO is known to efficiently produce ROS because of its high iron content. These results suggest that oxidant generation by the amphibole fibers likely contributes to the toxic nature of the amphibole particles, but it is probably not the only, or even the main, determinant.

One physical property that is noticeably different between the two size-fractionated samples is the particle length distribution. AM2.5 was found on average to consist of longer particles compared with LA2.5 (6.9 ± 12 μm vs. 1.9 ± 2.1 μm). The physical dimensions of asbestos minerals have been implicated in the mechanism of asbestos-induced inflammation in alveolar macrophages because of incomplete or “frustrated” phagocytosis of longer fibers (O'Neill, 2008); however, the effect of particle length on the relative toxicity of these amphibole minerals in epithelial cells grown in vitro is not as clear. Uptake of particles by epithelial cells is known to be a size-dependent process and may be a potential determinant that differentiates these two samples. Internalization of both the LA2.5 and AM2.5 particles was observed as early as 4 h after particle exposure by confocal microscopy (Fig. 5), and by 24 h, the particles were found localized primarily around the nucleus. Although no difference in particle uptake by the epithelial cells was apparent between the two fractionated samples using this technique, other factors including particle size distribution, particle number, and the mineralogical characteristics of the internalized particles could all potentially influence the cellular stress response. Current work is aimed at investigating these various factors and how they relate to the observed difference in proinflammatory response between the size-fractionated samples.

The relative toxicity of the different amphibole samples was initially assessed in this study by comparing the gene expression of the proinflammatory and stress-responsive genes IL-8, COX-2, and HO-1 that are known to be affected in these airway epithelial cells following insult by exposure to numerous inhaled particles. However, to better understand the cellular pathways affected in response to different amphibole types and different size fractions, a more detailed analysis of toxicity and stress pathways was carried out. This analysis included 84 genes involved in seven stress-responsive pathways. Similar to the results obtained previously in the more focused analysis of IL-8 and COX-2 expression, AM2.5 showed a different inflammatory profile than LA2.5 in these RT-PCR arrays. Although both showed increased production of transcripts coding for several proinflammatory cytokines such as TNF, increases in IL-1, IL-18, and IL-6 mRNA were limited to AM2.5. The elevation of both IL-1β and IL-18 transcripts is consistent with the activation of the inflammasome, a multiprotein complex responsible for initiation of inflammation and cell pyroptosis. NALP3, a member of the Nod-like receptor family, can form an inflammasome that has been proposed as a sensor for danger signals including oxidative and metabolic stress (Ogura et al., 2006; Schroder et al., 2010). Activation of the NALP3 inflammasome will activate caspase-1, which in turn will cleave the precursor proteins pro-IL-1β and pro-IL-18 to their active forms. This NALP3 inflammasome is known to be activated by ROS induction by silica and has now been shown to be central to the formation of silicosis. The ability of asbestos to activate the inflammasome is supported by Dostert et al. (2008), who showed that asbestos inhalation caused reduced inflammation (cell influx, cytokine production) in Nalp3(−/−) mice compared with wild type. Cassel et al. (2008) showed that asbestos-induced IL-1β production by lipopolysaccharide-stimulated macrophages is NALP3 dependent. Whether a similar process occurs in epithelial cells exposed to amphibole particles is unknown. Current work is aimed at examining more global patterns of gene expression changes utilizing microarray technology to obtain added insight into the mechanistic differences between these amphibole size fractions.

Asbestosis is a fibrotic pulmonary disorder that is present in increased prevalence in the Libby community. Perdue and Brody (1994) have shown in short-term rat inhalation studies that acute inflammation and the subsequent release of peptide growth factors occur primarily at the junction of the terminal bronchioles and alveolar ducts following exposure to chrysotile asbestos and that these events are implicated in the initiation of the fibrogenic response. The sequence of events that lead to a fibrotic phenotype have been shown to progress from the initiation of an inflammatory response to the subsequent release of growth factors followed by a fibroproliferative response by epithelial cells and the eventual stimulation of fibroblasts to release collagen resulting in the remodeling of the extracellular matrix. We show here that epithelial cells respond to amphibole particles with a dramatic increase in a number of proinflammatory cytokines. Continued production of these cytokines could lead to the production of growth factors such as TGF-β1, TGF-α, and platelet-derived growth factor (PDGF)-A and PDGF-B. Therefore, we examined the relative abundance of the mRNA transcripts encoding TGF-β1 in airway epithelial cells following exposure to AM2.5 and LA2.5 (Supplementary fig. S5). We saw no significant increase in mRNA transcript levels for this profibrotic factor after a 24-h exposure. This may be because a 24-h exposure is not long enough for growth factor gene expression to be increased or that the epithelial cell–produced cytokines stimulate production of growth factors by other cells such as alveolar macrophages.

In summary, we report here that water-elutriated, small size fractions of Libby amphibole and amosite (LA2.5 and AM2.5, respectively), which presumably penetrate deeper into the lung, are at least as potent in stimulating epithelial cell production of proinflammatory cytokines as unfractionated LA and AM. Furthermore, AM2.5 induced a 4- to 10-fold greater proinflammatory response compared with LA2.5. This difference in observed toxicity for the size-fractionated samples could not be explained by differences in mineral contamination between the two samples, TSA, or oxidant generation by the samples. We also showed that these small amphibole particles could be taken up by primary HAECs. Because the LA2.5 sample contains on average smaller amphibole particles than the AM2.5 sample, we hypothesize that differences in toxicity might be attributed to differential uptake of particles or to differences in the way the cell responds to internalized particles from each amphibole sample.

The present study provides evidence of the ability of Libby amphibole to induce epithelial cell injury and inflammation compared with other well-characterized amphibole samples. Our finding of a difference in toxicity between unfractionated and size-fractionated amphibole samples is of potential significance because this class of asbestiform minerals has a wide range of particle size distributions, some of which are small enough that they would be predicted to deposit deep in the lung. This study is one of the first studies to provide information about the contribution of a smaller “respirable” size fraction of asbestos particles, an area which was emphasized as an important data gap in the NIOSH Research Roadmap on Asbestos Fibers and Other Elongate Mineral Particles.

The findings presented here, although promising, are also preliminary. They need to be repeated using cells from additional donors, a wider concentration response curve, and additional asbestos samples. Further in vitro studies utilizing other relevant cell types and end points will also be required before an accurate assessment regarding the toxicity of LA can be firmly established. For example, these data do not address the issue of whether different size fractions have different genotoxic potential nor do they take into account the important role played by alveolar macrophages in interacting with inhaled asbestos. Perhaps the largest uncertainty associated with in vitro approaches is knowing whether they reflect changes that occur following in vivo exposures of animals or humans. Padilla-Carlin et al. (2010) found very similar comparative potencies to what we report here when they instilled into rats the same size-fractionated particles as were used in the current study. This coherence between in vitro and in vivo findings increases the confidence in the accuracy of the data as it translates to the predicted responses of this cell type in vivo.

Although in vitro toxicity assays have recognizable limitations, they are invaluable in their ability to rapidly screen numerous samples at relatively low cost. A recent NRC (2007) Report entitled “Toxicity Testing in the 21st Century: A Vision and a Strategy” recognized that it is increasingly possible to study the effects of toxicants using cultured cells, preferably of human origin, rather than whole animals. The report emphasized that in vitro toxicological evaluations using new methodologies that can rapidly assess global changes in mRNA expression and underlying toxicity pathways will be a vital tool for future toxicity testing using comparative approaches. We believe the data presented in this paper show the utility of this type of approach.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

U.S. Environmental Protection Agency (U.S. EPA) cooperative agreement (CR83346301); U.S. EPA Region 8, Denver, Libby Funds; U.S. EPA Office of Solid Waste and Emergency Response and Office of Superfund Remediation and Technology Innovation.

The authors gratefully acknowledge Greg Meeker and Heather Lowers (United States Geological Survey, Denver, CO) for their assistance in the SEM characterization of the asbestos samples. We thank Dr David Diaz-Sanchez (U.S. Environmental Protection Agency, Research Triangle Park, NC) and the members of the Libby Action Plan for helpful discussions.

References

Agency for Toxic Substances and Disease Registry (ATSDR)
Health Consultation: Mortality from Asbestosis in Libby, Montana, 1979–1998
 , 
2002
 
ATSDR, Atlanta, GA. Available at: http://www.atsdr.cdc.gov/asbestos/sites/libby_montana/mortality_review.html. Accessed October 5, 2010
Asgharian
B
Yu
CP
Deposition of inhaled fibrous particles in the human lung
J. Aerosol Med.
 , 
1988
, vol. 
1
 (pg. 
37
-
50
)
Becker
S
Mundandhara
S
Devlin
RB
Madden
M
Regulation of cytokine production in human alveolar macrophages and airway epithelial cells in response to ambient air pollution particles: further mechanistic studies
Toxicol. Appl. Pharmacol.
 , 
2005
, vol. 
207
 (pg. 
269
-
275
)
Blake
DJ
Bolin
CM
Cox
DP
Cardozo-Pelaez
F
Pfau
JC
Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages
Toxicol. Sci.
 , 
2007
, vol. 
99
 (pg. 
277
-
288
)
Borggaard
OK
Stucki
JW
Goodman
BA
Schwertmann
U
Phase Identification by Selective Dissolution Techniques
 , 
1988
Boston, MA
Reidel
(pg. 
83
-
98
)
Brody
AR
Hill
LH
Adkins
JB
O'Connor
RW
Chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages
Am. Rev. Respir. Dis.
 , 
1981
, vol. 
123
 (pg. 
670
-
679
)
Brody
AR
Roe
MW
Deposition pattern of inorganic particles at the alveolar level in the lungs of rats and mice
Am. Rev. Respir. Dis.
 , 
1983
, vol. 
128
 (pg. 
724
-
729
)
Brody
AR
Warheit
DB
Chang
LY
Roe
MW
George
G
Hill
LH
Initial deposition pattern of inhaled minerals and consequent pathogenic events at the alveolar level
Ann. N. Y. Acad. Sci.
 , 
1984
, vol. 
428
 (pg. 
108
-
120
)
Cassel
SL
Eisenbarth
SC
Iyer
SS
Sadler
JJ
Colegio
OR
Tephly
LA
Carter
AB
Rothman
PB
Flavell
RA
Sutterwala
FS
The Nalp3 inflammasome is essential for the development of silicosis
Proc. Natl. Acad. Sci. U.S.A.
 , 
2008
, vol. 
105
 (pg. 
9035
-
9040
)
Dai
YT
Yu
CP
Alveolar deposition of fibers in rodents and humans
J. Aerosol Med.
 , 
1998
, vol. 
11
 (pg. 
247
-
258
)
Dodson
RF
Atkinson
MAL
Levin
JL
Asbestos fiber length as related to potential pathogenicity: a critical review
Am. J. Ind. Med.
 , 
2003
, vol. 
44
 (pg. 
291
-
297
)
Donaldson
K
Borm
PJA
Oberdorster
G
Pinkerton
KE
Stone
V
Tran
CL
Concordance between in vitro and in vivo dosimetry in the proinflammatory effects of low-toxicity, low-solubility particles: the key role of the proximal alveolar region
Inhal. Toxicol.
 , 
2008
, vol. 
20
 (pg. 
53
-
62
)
Dostert
C
Petrilli
V
Van Bruggen
R
Steele
C
Mossman
BT
Tschopp
J
Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica
Science
 , 
2008
, vol. 
320
 (pg. 
674
-
677
)
Duvall
RM
Norris
GA
Dailey
LA
Burke
JM
McGee
JK
Gilmour
MI
Gordon
T
Devlin
RB
Source apportionment of particulate matter in the U.S. and associations with lung inflammatory markers
Inhal. Toxicol.
 , 
2008
, vol. 
20
 (pg. 
671
-
683
)
Frampton
MW
Ghio
AJ
Samet
JM
Carson
JL
Carter
JD
Devlin
RB
Effects of aqueous extracts of PM10 filters from the Utah Valley on human airway epithelial cells
Am. J. Physiol.
 , 
1999
, vol. 
277
 (pg. 
L960
-
L967
)
Ghio
AJ
Kennedy
TP
Whorton
AR
Crumbliss
AL
Hatch
GE
Hoidal
JR
Role of surface complexed iron in oxidant generation and lung inflammation induced by silicates
Am. J. Physiol.
 , 
1992
, vol. 
263
 (pg. 
L511
-
L518
)
Ghio
AJ
Zhang
J
Piantadosi
CA
Generation of hydroxyl radical by crocidolite asbestos is proportional to surface [Fe3+]
Arch. Biochem. Biophys.
 , 
1992
, vol. 
298
 (pg. 
646
-
650
)
Gozzelino
R
Jeney
V
Soares
MP
Mechanisms of cell protection by heme oxygenase-1
Annu. Rev. Pharmacol. Toxicol.
 , 
2010
, vol. 
50
 (pg. 
323
-
354
)
Graff
DW
Schmitt
MT
Dailey
LA
Duvall
RM
Karoly
ED
Devlin
RB
Assessing the role of particulate matter size and composition on gene expression in pulmonary cells
Inhal. Toxicol.
 , 
2007
, vol. 
19
 (pg. 
23
-
28
)
Halliwell
B
Gutteridge
JM
Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts
FEBS Lett.
 , 
1981
, vol. 
128
 (pg. 
347
-
352
)
Horton
K
Kapil
V
Larson
T
Muravov
O
Melnikova
N
Anderson
B
A review of the federal government's health activities in response to asbestos-contaminated ore found in Libby, Montana
Inhal. Toxicol.
 , 
2006
, vol. 
18
 (pg. 
925
-
940
)
Ilavska
S
Jahnova
E
Tulinska
J
Horvathova
M
Dusinska
M
Wsolova
L
Kyrtopoulos
SA
Fuortes
L
Immunological monitoring in workers occupationally exposed to asbestos
Toxicology
 , 
2005
, vol. 
206
 (pg. 
299
-
308
)
Institute of Medicine and the National Research Council of the National Academies: Committee for the Review of the NIOSH Research Roadmap on Asbestos Fibers and Other Elongate Mineral Particles
A Review of the NIOSH Roadmap for Research on Asbestos Fibers and Other Elongate Mineral Particles
 , 
2009
Washington, DC
The National Academies Press
Kamp
DW
Graceffa
P
Pryor
A
Witzman
SA
The role of free radicals in asbestos-induced diseases
Free Radic. Biol. Med.
 , 
1992
, vol. 
12
 (pg. 
293
-
315
)
Kennedy
TP
Dodson
RF
Rao
NV
Ky
H
Hopkins
C
Baser
M
Tolley
E
Hoidal
JR
Dusts causing pneumoconiosis generate .OH and produce hemolysis by acting as Fenton catalysts
Arch. Biochem. Biophys.
 , 
1989
, vol. 
269
 (pg. 
359
-
364
)
Kim
CS
Deposition of aerosol particles in human lungs: in vivo measurement and modelling
Biomarkers
 , 
2009
, vol. 
14
 (pg. 
54
-
58
)
Lang
DS
Schocker
H
Hockertz
S
Effects of crocidolite asbestos on human bronchoepithelial-dependent fibroblast stimulation in coculture: the role of IL-6 and GM-CSF
Toxicology
 , 
2001
, vol. 
159
 (pg. 
81
-
98
)
Lappi-Blanco
E
Kaarteenaho-Wiik
R
Maasilta
PK
Anttila
S
Paakko
P
Wolff
HJ
COX-2 is widely expressed in metaplastic epithelium in pulmonary fibrous disorders
Anat. Pathol.
 , 
2006
, vol. 
126
 (pg. 
717
-
724
)
Leyva
FJ
Roberts
K
Crocidolite induces prostaglandin I2 release mediated by vitronectin receptor and cyclooxygenase-2 in lung cells
Lung
 , 
2010
, vol. 
188
 (pg. 
133
-
141
)
Lippmann
M
Effects of fiber characteristics on lung deposition, retention, and disease
Environ. Health Perspect.
 , 
1990
, vol. 
88
 (pg. 
311
-
317
)
Lowers
HA
Bern
AM
Particle Size Characterization of Water-Elutriated Libby Amphibole 2000 and RTI International Amosite
2009
 
U.S. Geological Survey Open-File Report. Available at: http://pubs.usgs.gov/of/2009/1242/. Accessed October 5, 2010
McDonald
JC
Harris
J
Armstrong
B
Mortality in a cohort of vermiculite miners exposed to fibrous amphibole in Libby, Montana
Occup. Environ. Med.
 , 
2003
, vol. 
61
 (pg. 
363
-
366
)
McGavran
PD
Brody
AR
Chrysotile asbestos inhalation induces tritiated thymidine incorporation by epithelial cells of distal bronchioles
Am. J. Respir. Cell Mol. Biol.
 , 
1989
, vol. 
1
 (pg. 
231
-
235
)
Meeker
GP
Bern
AM
Brownfield
IK
Lowers
HA
Sutley
SJ
Hoefen
TM
Vance
JS
The composition and morphology of amphiboles from the Rainy Creek complex, near Libby, Montana
Am. Mineral.
 , 
2003
, vol. 
88
 (pg. 
1955
-
1969
)
Mills
PR
Davies
RJ
Devalia
JL
Airway epithelial cells, cytokines, and pollutants
Am. J. Respir. Crit. Care Med.
 , 
1999
, vol. 
160
 (pg. 
S38
-
S43
)
Mitchell
JA
Belvisi
MG
Akarasereenont
P
Robbins
RA
Kwon
O-J
Croxtall
J
Barnes
PJ
Vane
JR
Induction of cyclo-oxygenase-2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone
Br. J. Pharmacol.
 , 
1994
, vol. 
113
 (pg. 
1008
-
1014
)
Molinelli
AR
Madden
MC
McGee
JK
Stonehuerner
JG
Ghio
AJ
Effect of metal removal on the toxicity of airborne particulate matter from the Utah valley
Inhal. Toxicol.
 , 
2002
, vol. 
14
 (pg. 
1069
-
1086
)
Murthy
S
Adamcakova-Dodd
A
Perry
SS
Tephly
LA
Keller
RM
Metwali
N
Meyerholz
DK
Wang
Y
Glogauer
M
Thorne
PS
Carter
AB
Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis
Am. J. Physiol. Lung Cell Mol. Physiol.
 , 
2009
, vol. 
297
 (pg. 
L846
-
L855
)
National Research Council (NRC)
Toxicity Testing in the Twenty-First Century: A Vision and a Strategy
 , 
2007
Washington, DC
National Academies Press
Nishimura
Y
Nishiike-Wada
T
Miura
Y
Otsuki
T
Iguchi
H
Long-lasting production of TGF-β1 by alveolar macrophages exposed to low doses of asbestos without apoptosis
Int. J. Immunopathol. Pharmacol.
 , 
2007
, vol. 
20
 (pg. 
661
-
671
)
O'Neill
LAJ
How frustration leads to inflammation
Science
 , 
2008
, vol. 
320
 (pg. 
619
-
620
)
Ogura
Y
Sutterwala
FS
Flavell
RA
The inflammasome: first line of the immune response in cell stress
Cell
 , 
2006
, vol. 
126
 (pg. 
659
-
662
)
Padilla-Carlin
DJ
Schladweiler
MCJ
Kodavanti
UP
Shannahan
JH
Bern
AM
Lowers
HA
Meeker
GP
Gavett
SH
Inflammation and growth factor mRNA expression in lung tissue of F344 rats exposed to Libby amphibole asbestos
Am. J. Respir. Crit. Care Med.
 , 
2010
, vol. 
181
 pg. 
A1756
  
(Abstract)
Park
EJ
Chung
KH
Ryu
DY
Choi
J
Park
K
Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells
Toxicol. Lett.
 , 
2008
, vol. 
180
 (pg. 
222
-
229
)
Perdue
TD
Brody
AR
Distribution of transforming growth factor-β1, fibronectin, and smooth muscle actin in asbestos-induced pulmonary fibrosis in rats
J. Histochem. Cytochem.
 , 
1994
, vol. 
42
 (pg. 
1061
-
1070
)
Rosenthal
GJ
Germolec
DR
Blazka
ME
Corsini
E
Simeonova
P
Pollock
P
Kong
L-Y
Kwon
J
Luster
MI
Asbestos stimulates IL-8 production from human lung epithelial cells
J. Immunol.
 , 
1994
, vol. 
153
 (pg. 
3237
-
3244
)
Roth
CB
Jackson
ML
Syers
JK
Deferration effect on structural ferrous-ferric iron ratio and CEC of micaceous vermiculites and soils
Clays Clay Miner.
 , 
1969
, vol. 
17
 (pg. 
253
-
264
)
Rouse
RL
Murphy
G
Boudreaux
MJ
Paulsen
DB
Penn
AL
Soot nanoparticles promote biotransformation, oxidative stress, and inflammation in murine lungs
Am. J. Respir. Cell Mol. Biol.
 , 
2008
, vol. 
39
 (pg. 
198
-
207
)
Samet
JM
Reed
W
Ghio
AJ
Devlin
RB
Carter
JD
Dailey
LA
Bromberg
PA
Madden
MC
Induction of prostaglandin H synthase 2 in human airway epithelial cells exposed to residual oil fly ash
Toxicol. Appl. Pharmacol.
 , 
1996
, vol. 
141
 (pg. 
159
-
168
)
Schroder
K
Zhou
R
Tschopp
J
The NLRP3 inflammasome: a sensor for metabolic danger?
Science
 , 
2010
, vol. 
327
 (pg. 
296
-
300
)
Setta
JH
Neder
JA
Bagatin
E
Terra-Filho
M
Napolis
LM
Corso
SD
Rodrigues
RT
Fernandes
ALG
Nery
LE
Relationship between induced sputum cytology and inflammatory status with lung structural and functional abnormalities in asbestosis
Am. J. Ind. Med.
 , 
2008
, vol. 
51
 (pg. 
186
-
194
)
Stanton
MF
Layard
M
Tegeris
E
Miller
E
May
M
Morgan
E
Smith
A
Relation of particle dimension to carcinogenicity in amphibole asbestoses and other fibrous minerals
J. Natl. Cancer Inst.
 , 
1981
, vol. 
67
 (pg. 
965
-
975
)
Sturm
R
Hofmann
W
A theoretical approach to the deposition and clearance of fibers with variable size in the human respiratory tract
J. Hazard. Mater.
 , 
2009
, vol. 
170
 (pg. 
210
-
218
)
Sullivan
PA
Vermiculite, respiratory disease, and asbestos exposure in Libby, Montana: update of a cohort mortality study
Environ. Health Perspect.
 , 
2007
, vol. 
115
 (pg. 
579
-
585
)
Tal
TL
Graves
LM
Silbajoris
R
Bromberg
PA
Wu
W
Samet
JM
Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor signaling in human airway epithelial cells exposed to Zn2+
Toxicol. Appl. Pharmacol.
 , 
2006
, vol. 
214
 (pg. 
16
-
23
)
Tal
TL
Simmons
S
Silbajoris
R
Dailey
LA
Cho
SH
Ramabhadran
R
Linak
W
Reed
W
Bromberg
PA
Samet
JM
Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles
Toxicol. Appl. Pharmacol.
 , 
2010
, vol. 
243
 (pg. 
46
-
54
)
U.S. Environmental Protection Agency
Dosimetric and Toxicologic Assessment of Amphibole Fiber-Containing Material from Libby, Montana
 , 
2007
 
Venkataraman
C
Comparison of particle lung doses from the fine and coarse fractions of urban PM-10 aerosols
Inhal. Toxicol.
 , 
1999
, vol. 
11
 (pg. 
151
-
169
)
Webber
JS
Blake
DJ
Ward
TJ
Pfau
JC
Separation and characterization of respirable amphibole fibers from Libby, Montana
Inhal. Toxicol.
 , 
2008
, vol. 
20
 (pg. 
733
-
740
)
Whitehouse
AC
Asbestos-related pleural disease due to tremolite associated with progressive loss of lung function: serial observations in 123 miners, family members, and residents of Libby, Montana
Am. J. Ind. Med.
 , 
2004
, vol. 
46
 (pg. 
219
-
225
)
Whitehouse
AC
Black
CB
Heppe
MS
Ruckdeschel
J
Levin
SM
Environmental exposure to Libby asbestos and mesotheliomas
Am. J. Ind. Med.
 , 
2008
, vol. 
51
 (pg. 
877
-
880
)
Wu
W
Samet
JM
Ghio
AJ
Devlin
RB
Activation of EGF receptor signaling in human airway epithelial cells exposed to ambient air particles
Am. J. Physiol. Lung Cell Mol. Physiol.
 , 
2001
, vol. 
281
 (pg. 
L483
-
L489
)

Author notes

Disclaimer: The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.