ToxSci Advance Access originally published online on June 19, 2007
Toxicological Sciences 2007 99(1):277-288; doi:10.1093/toxsci/kfm166
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Internalization of Libby Amphibole Asbestos and Induction of Oxidative Stress in Murine Macrophages
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* Division of Biological Sciences and Department of Biomedical & Pharmaceutical Sciences
Center for Environmental Health Sciences, University of Montana, Missoula, Montana 59812
1 To whom correspondence should be addressed at Center for Environmental Health Sciences, University of Montana, Missoula, MT 59812. Fax: (406) 243-2807. E-mail: jean.pfau{at}umontana.edu.
Received April 4, 2007; accepted June 14, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.
Key Words: asbestos; Libby amphibole; murine macrophage; oxidative stress; DNA damage.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Asbestos exposure in humans is associated with the development of asbestos-related diseases (ARD) such as pulmonary fibrosis and lung cancer. Although the exact mechanism leading to the progression of ARD has not been fully explained, mounting evidence indicates that reactive oxygen species (ROS), such as the superoxide anion and the hydroxyl radical, plays a significant role (Bhattacharya et al., 2005
; Kamp and Weitzman, 1999). Because airways are continuously exposed to high levels of environmental oxidants, they must maintain the proper balance between pro-oxidants and antioxidants to prevent oxidative stress and cellular damage. Environmental toxicants that alter the cellular redox state in the lung promote oxidative stress and lead to pulmonary injury. Oxidative stress is therefore associated with numerous pulmonary diseases including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis (Rahman et al., 2006).
The vermiculite mine and surrounding community of Libby, MT, were designated as EPA Superfund sites in 2002 due to the asbestos contamination of vermiculite, which led to significant asbestos exposure throughout the mine site and surrounding area (Wright et al., 2002). The extensive asbestos exposure has led to considerable health problems in the community including reduced pulmonary function, enhanced autoimmune responses, and increased mortality from lung cancer, malignant mesothelioma, and fibrosis (McDonald et al., 2004; Pfau et al., 2005; Whitehouse, 2004). The form of asbestos in Libby's contaminated vermiculite has been characterized in the amphibole family of fibers and consists of several hydrated silicate fibers, including regulated fibers (tremolite) and unregulated fibers (winchite and richterite) (Meeker et al., 2003). The regulated and unregulated fibers within Libby amphibole asbestos differ in terms of length and in the metallic cations expressed on their surface. Because of their diverse chemical composition Libby amphibole fibers are different from other well-studied amphibole fibers, such as crocidolite and amosite, and therefore may induce distinct cellular effects in exposed cells.
Crocidolite asbestos generates an increase in ROS, which leads to a depletion of intracellular glutathione (GSH) and oxidative damage to DNA and lipids in several cell types (Faux and Howden, 1997; Fung et al., 1997; Golladay et al., 1997; Janssen et al., 1992; Kamp et al., 1992; Kim et al., 2001; Vallyathan et al., 1992; Xu et al., 2002; Yamaguchi et al., 1999). Moreover, ROS induced by crocidolite plays an important role in the activation of redox sensitive transcription factors such as nuclear factor-kappa B and activator protein 1 (Faux and Howden, 1997; Flaherty et al., 2002). Because ROS generated by amphibole asbestos results in oxidative stress, the mechanism by which asbestos induces ROS production has been extensively studied. However, the exact source of ROS in response to asbestos fibers is an area of considerable debate and may be due to the unique chemical characteristics of the asbestos fiber (Kamp and Weitzman, 1999).
Amphibole asbestos generates increased levels of ROS through at least two independent mechanisms. Amphibole asbestos fibers, specifically crocidolite and amosite, participate in the direct production of ROS via iron-catalyzed reactions due to their high iron content (Kamp and Weitzman, 1999; Mossman and Gee, 1997). This mechanism is supported by studies demonstrating that crocidolite asbestos cytotoxicity can be abrogated in the presence of iron chelators (Goodglick and Kane, 1986). The importance of iron in asbestos fiber chemistry is also supported in vivo given the fact that iron chelators protect against pulmonary inflammation and fibrosis (Kamp et al., 1995). The second proposed mechanism by which amphibole asbestos generates oxidative stress is through the production of mitochondrial-derived ROS. Alveolar epithelial cells generate intracellular ROS, which results in DNA damage and apoptosis in response to amosite asbestos (Panduri et al., 2004, 2006).
The alveolar macrophage is the primary cell type that interacts with inhaled particles and functions to clear particles from the lung. Therefore, our study utilizes primary murine alveolar macrophages and a well-characterized macrophage cell line. Our data indicate that Libby amphibole asbestos induces oxidative stress in murine macrophages through increasing ROS levels and suppressing superoxide dismutase (SOD) activity. The cellular effects of Libby asbestos exposure appear to be distinct from what is observed with crocidolite asbestos and may be a result of the activation of separate cellular mechanisms within exposed macrophages. This is the first study to elucidate the cellular changes in macrophages as a result of exposure to Libby amphibole asbestos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Cell culture conditions.
Mouse macrophages, RAW264.7 cells (ATCC-2091: American Type Culture Collection, Manassas, VA) were cultured at 37°C in a 5% CO2 incubator (Thermo Forma, Waltham, MA) in complete media, which contained Dulbecco's modified Eagle's medium media (cDMEM) with 4.5 g/l glucose and L-glutamine supplemented with 1.5mM sodium pyruvate, 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 55µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B) (Gibco BRL, Bethesda, MD). Confluent RAW264.7 cells were scraped from T75 flasks, counted with a Z series Coulter Counter (Beckman Coulter, Hialeah, FL), and plated into 96-, 12-, and 6-well plates or T75 flasks in complete media and allowed to adhere overnight prior to exposure to asbestos fibers. Primary alveolar macrophages were lavaged from C57BL/6 mice as previously described (Migliaccio et al., 2005
), plated, and immediately exposed to asbestos. To elucidate the role of free radical scavengers, cells were incubated with SOD coupled to methoxypolyethylene glycol (PEG) overnight at a final concentration of 19.6 Units (U) per ml (Sigma Chemical Co., St Louis, MO), and concomitantly exposed to asbestos fibers. All experiments were performed using adherent cells only.
Particulate matter.
Three types of fibers were used in this study. Libby asbestos was obtained from the U.S. Geological Survey. The Libby amphibole fibers have been chemically and physically characterized in detail (Gunter et al., 2003; Meeker et al., 2003; Wylie and Verkouteren, 2000). The Libby asbestos sample is chemically representative of the amphibole in the mine and has a particle size distribution that matches the air sample size distribution data (Meeker et al., 2003). Libby asbestos contains six different amphibole fiber types, therefore, the asbestos sample is labeled in this paper as 6-mix. Wollastonite, a noncytotoxic, nonfibrogenic control fiber, was provided by NYCO Minerals (Willsboro, NY). Crocidolite asbestos was provided by the Research Triangle Institute (RTI, NC). All fibers were dispersed in phosphate-buffered saline (PBS, pH 7.4) by cup-horn sonication (Misonix, Framingdale, NY) before culturing. Stock concentration suspensions of fibers were prepared fresh immediately prior to their addition into cDMEM cell cultures. Fiber concentrations were based on relative mass. The size distributions of all three fiber types are presented in Table 1.
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Transmission electron microscopy.
RAW264.7 cells were exposed to Libby asbestos fibers at a concentration of 5 µg/cm2 for 24 h. Cells were washed three times in PBS, detached with a cell scraper, and fixed with 4% glutaraldehyde in PBS for 4 h at 25°C. Cells were centrifuged for 5 min at 1000 x g, washed three times in PBS, and the pellet was suspended in low melt agarose. The pellet was postfixed in 1% osmium tetraoxide in PBS (pH 7.4) for 1 h at 25°C, and washed three times in PBS. The pellet was dehydrated in ethanol and embedded in resin. Ultrathin sections (50 nm) were examined under a Hitachi H-7100 transmission electron microscopy (Hitachi, Ibaraki, Japan) with a tungsten filament and captured with an Advanced Microscopy Techniques digital camera (AMT, Danvers, MA). Sections were viewed at 75 kV. Images were captured with the AMT software version 540.
Cell viability.
RAW264.7 cells were exposed to fiber concentrations and duration stated in the figures. Cells were incubated with trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco BRL) for 5 min at room temperature. Complete media were added to stop the reaction. The cells were washed once and centrifuged at 1000 x g for 10 min. The pellet was resuspended in 200 µl of PBS to obtain a single cell suspension. Cells were permeabilized with 1 ml of 80% ethanol and incubated at –20°C overnight. Cells were washed once in PBS, and DNA was stained with 1 ml of PBS containing 0.5% Triton X-100 (Sigma), 50 µg/ml RNase A (Roche, Indianapolis, IN), and 50 µg/ml propidium iodide (PI) (Invitrogen, Carlsbad, CA), and then incubated for 40 min at 37°C. Cells were transferred to filter-top polypropylene tubes (BD Labware, Franklin Lakes, NJ) and stored on ice for analysis using the FACS Caliber (BD Biosciences, San Jose, CA) for red fluorescence. Dead and apoptotic cells were identified in the sub-G0/G1 peak determined by PI immunofluorescence. Cell viability was calculated as the percent of viable cells (cells above the sub-G0/G1 phase) divided by the total percent of cells and expressed as percent of control.
Cell viability of RAW264.7 cells after 3 h of exposure to Libby asbestos at a final concentration of 62.5 µg/cm2 was not significantly different than controls as determined by a one-way ANOVA. These results were confirmed by a lactate dehydrogenase cytotoxicity assay (BioVision, Mountain View, CA) and the CellTiter-Blue Reagent assay (Promega, Madison, WI).
Enumeration of internalized fibers.
Fiber uptake was quantified as previously described with slight modifications (Boylan et al., 1995). Briefly, RAW264.7 cells were exposed to Libby asbestos for 3 h at a final concentration of 62.5 µg/cm2. Cells were washed with PBS and detached with trypsin-EDTA (Gibco) for 10 min with gentle rotation at room temperature to remove any fibers that were adherent but not internalized. Complete media were added to stop the reaction. The cells were washed in PBS and then examined under phase contrast microscopy to count the number of internalized fibers. The number of fibers per cell was quantified from 0 to 5. Greater than five fibers per cell were impossible to differentiate, therefore, the upper limit of detection was five fibers per cell. Cells with more than five internalized fibers were classified into the upper limit (5). Fifty cells in random fields were enumerated for each treatment. The experiment was performed twice with similar results.
Quantification of ROS in response to Libby amphibole asbestos.
ROS production was measured using dichlorofluorescein diacetate (DCFDA, Molecular Probes, Eugene, OR) and dihydroethidine (DHE, Molecular Probes). 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) crosses the cell membrane and is trapped in the cell after deacetylation by intracellular esterases to 2,7-dichlorodihydrofluorescein (DCFH). DCFH is then sensitive to oxidation, forming the fluorescent compound dichlorofluorecein (Halliwell and Whiteman, 2004). DHE is more specific for superoxide than DCFDA and is also oxidized to a fluorescent product (Zhao et al., 2003). Cells were plated in 96-well plates and incubated overnight at 37°C and 5% CO2. Cells were incubated with 40 µM DCFDA for 1 h or in 2µM DHE for 30 min in a 37°C and 5% CO2 incubator then exposed to fibers at concentrations stated in the figures. The plate was returned to the 37°C, 5% CO2 incubator between hourly readings on the fluorescent plate reader. Cells without dye were used to subtract background fluorescence. Readings for fluorescence intensity were measured using a SpectraMax fluorescent plate reader set at 485-nm excitation and 530-nm emissions for DCFDA quantification and 518-nm excitation and 605-nm emission for DHE quantification (Molecular Devices, Sunnyvale, CA).
Quantification of total SOD activity.
Total SOD activity was measured as described previously (Cardozo-Pelaez et al., 1998). Briefly, RAW264.7 cells were exposed to asbestos for 3, 7, 12, and 24 h at a final concentration of 62.5 µg/cm2. The cells were washed with PBS and homogenized in 10mM EDTA disodium salt buffer (pH 8.0) by cup-horn sonication. Samples were centrifuged at 20,000 x g at 4°C for 15 min. The supernatants (15 µl) were incubated for 20 min in a 25°C water bath with 150 µl phosphate buffer, 15 µl of 1.5mM xanthine, and 15 µl of 1.0mM hydroxylamine chloride. The reaction was initiated by the addition of 75 µl of 0.8 mg protein/ml xanthine oxidase (Sigma). An aliquot of the reaction (100 µl) was added to a mixture of 100 µl of 19mM sulfanilic acid and 100 µl of 7mM 
-napthylamine in a 96-well plate, incubated at room temperature for 20 min, and the absorbance was read at 539 nm using a SpectraMax plate reader (Molecular Devices). The activity of total SOD was determined from a standard curve using known amounts of purified SOD and quantified as Units of SOD activity per milligram of protein. Data are expressed as the percent of total SOD activity compared to time-matched controls.
Quantification of reduced GSH.
GSH levels were measured using the GSH recycling assay as previously described (Schneider et al., 2005). RAW264.7 cells were exposed to asbestos at concentrations and durations stated in the figures. The cells were washed with PBS and lysed with two freeze/thaw cycles in 300 µl of 10mM HCl. Fifty-microliter aliquots were taken for protein determination. Protein was precipitated by adding 70 µl of 6.5% sulfosalicylic acid, incubating on ice for 10 min, and centrifuging at 2000 x g for 15 min. Two hundred microliters of supernatant was transferred to a 96-well plate for GSH quantification. Total intracellular GSH was measured using the GSH reductase-5,5'- dithiobis(2-nitrobenzoic acid) recycling assay, comparing the rate of color formation at 412 nm of the unknowns to a standard curve. Total intracellular GSH levels were determined by quantifying the intracellular GSH levels and dividing by the protein concentration to obtain nanomoles of GSH per milligram (mg) protein. Data are expressed as the percent of total GSH levels compared to time-matched controls.
Quantification of 8-hydroxy-2'-deoxyguanosine.
RAW264.7 cells were exposed to asbestos for 12 and 24 h and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) levels were quantified using the method previously described (Bolin et al., 2004). Briefly, DNA was isolated through phenol-chloroform extraction and digested with nuclease P1 (Roche). 8-oxo-dG and 2-deoxyguanosine (2dG) were resolved by high-performance liquid chromatography with a reverse phase YMCBasic column (YMC, Inc., Wilmington, NC) and quantified using a CoulArray electrochemical detection system (ESA, Inc., Chelmsford, MA). Calibration curves were generated from standards of 2-dG (Sigma) ranging from 100 ng to 2 µg and 8-oxo-dG (Cayman Chemical, Ann Arbor, MI) ranging from 5 to 100 pg. The amount of 2-dG and 8-oxo-dG in control and treated cells was calculated according to the calibration curves. The relative levels of 8-oxo-2dG are expressed as the ratio of 8-oxo-2dG (fmol)/2dG (nmol). Data were recorded, analyzed, and stored using CoulArray for Windows data analysis software.
Determination of DNA damage.
DNA damage was determined by the comet assay according to previously published methods with minor modifications (Tice et al., 1991). Briefly, 5 µl of the cell suspension was embedded in 75 µl of 0.5% low melt agarose (Bio-Rad, Hercules, CA) and sandwiched between a layer of 5 µl of 0.5% normal melting agarose and a top layer of 0.5% low melting agarose on conventional microscope slides. To lyse cellular and nuclear membranes of the embedded cells, slides were immersed in ice-cold, freshly prepared, lysis solution (2.5M NaCl, 100mM disodium EDTA, 10mM Tris–Cl, and 10% dimethyl sulfoxide [DMSO], pH 13.0) and incubated at 4°C for 1 h. The slides were incubated in alkaline buffer (0.1% 8-hydroxyquinoline, 10mM disodium EDTA, 2% DMSO, and 200mM NaOH) for 20 min to allow DNA unwinding. Electrophoresis was performed in alkaline buffer at 300 mA for 20 min. After electrophoresis, the slides were neutralized with 400mM Tris–Cl, pH 7.4 twice for 20 min. DNA was stained with 100 µl per slide of Hoechst (10 µg/ml) (Molecular Probes). All steps were conducted in darkness to prevent additional DNA damage and samples were analyzed within 4 h.
Microscopic analysis.
A laser scanning cytometer (LSC, Compucyte, Cambridge, MA) equipped with a BX50 Olympus microscope and a 407-nm argon laser was used to scan the slides. Hoechst fluorescence was detected with a photomultiplier tube equipped with a 460- to 485-nm bandpass filter. The embedded cells were focused and scanned on the central portion of each well. The sensitivity threshold was set to contour comet heads only. A threshold value of greater than 3000 provided a good discrimination between nuclear fluorescence and comet tails. Fluorescence was integrated from a region of six pixels broader than the threshold around the comet head as previously described (Petersen et al., 2000). For each integration contour, the integrated fluorescence, maximum pixel, and the area were collected. The scanning was run by using a x 20 dry objective. Two thousand events were scanned per slide. A histogram of integral fluorescence was compared to that obtained by flow cytometric cell cycle analysis through LSC software (WinCyte, Compucyte). The G1 and G2 phase peak were confirmed by LSC imaging. Cells with damaged DNA contained less DNA and stained with lower fluorescence intensity than the G1 phase cells and therefore appeared in the sub-G1 area of the histogram.
Quantification of 8-oxoguanine-DNA-glycosylase 1 (Ogg1) activity.
RAW264.7 cells were exposed to asbestos for 3, 7, and 12 h and Ogg1 activity levels were quantified using the method previously described (Bolin et al., 2004). Briefly, DNA glycosylase was extracted from the pellets of the EDTA sample buffers, as described above, through homogenization at 4°C in extraction buffer containing 20mM Trizma-base (pH 8.0), 1mM EDTA, 0.5mM spermine, 0.5mM spermidine, 1mM dithiotreitol, 50% glycerol, and protease inhibitor cocktail (Roche). Following the addition of 2.5M potassium chloride, the homogenate was incubated at 4°C for 30 min. Aliquots of the supernatant were collected following centrifugation at 20,000 x g for 30 min and stored at – 80°C. Ogg1 activity was determined using a synthetic probe containing 8-oxo-dG (Trevigen, Gaithersburg, MD) labeled with 
-32P at the 5'-end, using T4 polynucleotide kinase (Roche). The probe used has the nucleotide sequence 5'-GAACTAGTGXATCCCCCGGGCTGC-3' (X = 8-oxo-dG) and was annealed to its corresponding complimentary oligonucleotide before the nicking reaction was preformed. The nicking reaction was initiated by incubating 2.5 µg of protein extract and the double-stranded probe for 30 min at 37°C and stopped by placing the samples on ice. Aliquots of loading buffer containing 90% formamide, 10mM NaOH, and blue–orange dye (Promega) were then added to each sample. After 5 min at 95°C, samples were chilled and loaded into a polyacrylamide gel (20%) with 7M urea and 1x Tris-Borate-EDTA and run at 400 V for 2 h. Gels were quantified using FLA-3000 Series Fuji Film Fluorescent Image Analyzer and analysis software. The capacity of the extract to remove 8-oxo-dG was expressed as a percentage of the cleaved synthetic probe to the total probe used in densitometric units.
Protein determination.
Protein concentrations were determined with the BCA (bicinchoninic acid) protein assay kit based on the BCA method (Pierce, Rockford, IL). The assay, adapted for microtiter plates, was used according to the manufacture's instructions.
Statistical analysis.
Data are given as mean ± SD or mean ± SEM. Analyses were done using the software package GraphPad Prism 3.03 (GraphPad, San Diego, CA). One-way or two-way ANOVA was used to compare groups with one independent or two independent variables, respectively. A Bonferroni or Dunnett's posttest was used to compare different treatments. Data comparing two group means were analyzed by independent samples t test. Significance was noted at p < 0.05 and adjusted for the number of comparisons according to Bonferroni's adjustment. Nonparametric analysis of fiber uptake was conducted using the Kruskal–Wallis test. Outliers were detected through Grubb's test from GraphPad Software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Libby Asbestos Fibers are Internalized by Murine Macrophages
To determine whether Libby asbestos fibers are internalized in vitro, murine macrophages were exposed to Libby asbestos at a concentration of 5 µg/cm2 for 24 h and analyzed through transmission electron microscopy. After 24 h of asbestos exposure, murine macrophages contained variable numbers of asbestos fibers, which were less than 2 µm in length. The majority of Libby asbestos fibers were observed in cytoplasmic vacuoles or protruding from cytoplasmic vacuoles into the cytosol (Figs. 1A and 1B, respectively), and localized primarily around the nucleus. Libby asbestos fibers were also observed attached to the plasma membrane (Fig. 1C) and free within the cytoplasm (Fig. 1D). These results indicate that murine macrophages phagocytize Libby asbestos fibers and the internalized fibers localize to cytoplasmic vacuoles and to the cytosol.
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Libby Asbestos Increases Intracellular ROS in Murine Macrophages
To establish whether macrophages produce increased levels of ROS in response to amphibole asbestos, RAW264.7 cells were incubated with 40µM DCFDA for 1 h then exposed to increasing concentrations of Libby asbestos, ranging from 6.25 to 62.5 µg/cm2. DCFDA is a fluorescent dye used to indirectly quantify the amount of intracellular ROS. Therefore, the relative fluorescence intensity is correlated to the amount of intracellular ROS. Fluorescent readings were taken every hour for 3 h. The relative fluorescence intensities in macrophages over time are shown in Figure 2A. Murine macrophages increased intracellular ROS levels in response to Libby asbestos in a dose-dependent manner. The lowest concentration of Libby asbestos (6.25 µg/cm2) significantly increased the relative fluorescence after 3 h of exposure compared to untreated cells (p < 0.05). However, higher concentrations of Libby asbestos significantly increased the relative fluorescence after only 1 h of exposure (p < 0.05). The highest concentration of Libby asbestos (62.5 µg/cm2) generated the greatest increase in ROS and this concentration of asbestos did not reduce cell viability within 3 h as determined by PI immunofluorescence (Fig. 2B) and two additional viability assays (see "Materials and Methods"). The viability of RAW264.7 cells after 3, 7, 12, and 24 h of exposure to Libby and crocidolite asbestos is shown in Figure 2B. The viability of RAW264.7 cells after 24 h of exposure to Libby and crocidolite asbestos was 92% and 62%, respectively, compared to untreated cells. Exposure to wollastonite fibers for 24 h did not reduce the viability of RAW264.7 cells compared to untreated controls (data not shown). Since the highest concentration of Libby asbestos induced the greatest increase in ROS without decreasing cell viability, we utilized this fiber concentration for all subsequent experiments.
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In order to determine whether the increase in ROS was unique to Libby asbestos, murine macrophages were exposed to equal concentrations of Libby asbestos, wollastonite fibers, a nonfibrogenic control fiber (Tatrai et al., 2004
), and a well-characterized cytotoxic asbestos fiber, crocidolite. As shown in Figure 2C, cells exposed to Libby asbestos had a significantly higher relative fluorescence after only 1 h of exposure compared to untreated cells (p < 0.05). Exposure to wollastonite fibers did not increase the level of fluorescence in macrophages at any time during exposure compared to control cells. Cells exposed to crocidolite increased the level of fluorescence after 3 h of exposure; however, the increase in ROS was lesser in magnitude compared to Libby asbestos exposure. Similar results were obtained using primary alveolar macrophages lavaged from C57BL/6 mice (data not shown). These results demonstrate that exposure to Libby and crocidolite asbestos increases intracellular ROS in murine macrophages and the increase in intracellular ROS occurs in primary alveolar macrophages as well as in macrophage cell line cells.
Equivalent Number of Murine Macrophages Interact and Internalize Crocidolite and Libby Asbestos Fibers
To determine whether the increase in ROS was dependent upon the number of cell-fiber interactions or fiber uptake, we used light microscopy to enumerate the number of cells interacting with asbestos fibers and the number of internalized fibers per cell. To this end, murine macrophages were exposed to equal concentrations of Libby and crocidolite asbestos and wollastonite fibers for 3 h as described above. Cells were washed extensively and examined under phase contrast light microscopy. The number of cells interacting with one or more fibers was enumerated for each treatment. One hundred cells were counted for each treatment.
The preparation of Libby asbestos fibers contained numerous fibers of different lengths and widths. As seen in Figure 3A, Libby asbestos fibers visualized through light microscopy were generally less than 100 µm in length. Additionally, all murine macrophages bound or internalized one or more Libby asbestos fibers after 3 h. The preparation of crocidolite asbestos was more homogeneous in morphology and contained longer fibers than the Libby asbestos. All murine macrophages also established multiple interactions with crocidolite asbestos fibers after 3 h (Fig. 3B). Wollastonite fibers interacted with 25% of macrophages and had fewer fibers in each preparation compared to either amphibole asbestos preparations (Fig. 3C). These results indicate that equal numbers of murine macrophages bind Libby and crocidolite asbestos fibers after 3 h at the concentrations used in this study.
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The number of internalized fibers per cell was quantified as previously described with minor modifications (Boylan et al., 1995
). On average, macrophages internalized 4.38 ± 1.06 Libby asbestos fibers per cell and 3.28 ± 1.58 crocidolite asbestos fibers per cell (mean ± SD). The difference between the numbers of Libby and crocidolite asbestos fibers internalized per cell was not significant. In contrast, macrophages internalized significantly fewer wollastonite fibers per cell (0.88 ± 0.93) compared to the number of amphibole asbestos fibers (p < 0.05). Together these data indicate that the increase in the level of ROS in macrophages is not dependent upon the number of cellular interactions with the asbestos fibers nor is it dependent on the number of internalized amphibole fibers.
Increased ROS Levels Generated by Libby Asbestos is Suppressed with the Addition of Exogenous Intracellular SOD
To determine whether ROS induced by Libby amphibole asbestos can be inhibited by a free radical scavenger, RAW264.7 cells were incubated overnight with SOD coupled to PEG. SOD catalyzes the formation of hydrogen peroxide from superoxide anion and the conjugation of PEG leads to the enhanced uptake of exogenous SOD into cells (Beckman et al., 1988). After treatment with PEG–SOD, macrophages were washed and subsequently exposed to Libby asbestos, therefore, the effect of PEG–SOD on asbestos-induced ROS production can only be attributed to intracellular SOD activity. As shown in Figure 4A, macrophages exposed to Libby asbestos had a significantly higher level of intracellular ROS after 3 h of asbestos exposure (p < 0.05). However, macrophages pretreated with PEG–SOD and subsequently exposed to Libby asbestos had significantly lower levels of intracellular ROS compared to macrophages exposed to asbestos alone (p < 0.05). Pretreatment of macrophages with PEG–SOD did not change the levels of ROS compared to control cells, indicating that the protective effect of PEG–SOD during asbestos exposure can be attributed to a reduction in superoxide levels. Pretreatment of cells with PEG alone did not inhibit ROS produced by Libby asbestos (data not shown). These data confirm that Libby asbestos-induced ROS is predominately intracellular and can be abrogated by PEG–SOD, suggesting that the superoxide anion is the major contributor to the increased ROS levels.
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Libby Asbestos Increases Superoxide Production in Murine Macrophages
To provide additional support for the premise that a contributing ROS generated by Libby amphibole asbestos is the superoxide anion, RAW264.7 cells were incubated with 2µM DHE for 30 min, and then exposed to Libby asbestos at a final concentration of 62.5 µg/cm2. DHE is a fluorescent dye used to indirectly quantify the amount of superoxide in cells. Therefore, the relative fluorescent intensity is correlated to intracellular superoxide levels (Zhao et al., 2003
). The relative DHE fluorescence intensity of murine macrophages exposed to Libby asbestos and wollastonite fibers is shown in Figure 4B. Macrophages exposed to Libby asbestos had a significantly higher relative fluorescence after 2 h of exposure compared to untreated cells (p < 0.05). Macrophages exposed to wollastonite fibers did not increase the level of fluorescence compared to control. These data indicate that Libby asbestos exposure increases intracellular levels of superoxide in murine macrophages.
Total Intracellular SOD Activity is Suppressed by Libby Amphibole Asbestos
Because Libby asbestos exposure increases superoxide levels in murine macrophages, we hypothesized that the increase in superoxide may be due to a modification in SOD activity. Therefore, total intracellular SOD activity was quantified in murine macrophages after 3, 7, 12, and 24 h of exposure as previously described (Cardozo-Pelaez et al., 1998). Libby asbestos suppressed total SOD activity by 30% after 3 h of exposure and the reduction in activity was significant compared to untreated controls (p < 0.05) (Fig. 5). However, total SOD activity was not significantly different compared to controls after 7, 12, and 24 h of exposure to Libby asbestos. Total SOD activity was not different in cells exposed to wollastonite compared to time-matched controls. Crocidolite asbestos significantly increased total SOD activity by 200% after 24 h of exposure compared to untreated controls (p < 0.05). These results indicate that exposure to Libby asbestos results in an initial suppression of total SOD activity and that exposure to crocidolite asbestos increases total SOD activity after 24 h in murine macrophages.
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Intracellular GSH Levels are Reduced in Response to Libby Amphibole Asbestos
In response to increased levels of ROS, cells utilize GSH to maintain the intracellular redox balance within cells. Reduced levels of intracellular GSH in cells are an indication of oxidative stress. Therefore, intracellular GSH was measured in macrophages after exposure to amphibole asbestos and wollastonite fibers to determine whether exposure results in a decrease in intracellular GSH levels. The intracellular GSH levels were measured through the GSH recycling assay as previously described (Schneider et al., 2005
). In response to Libby asbestos, macrophages significantly decreased intracellular GSH levels compared to untreated cells at 7, 12, and 24 h (p < 0.05) (Fig. 6). Exposure to crocidolite asbestos also significantly decreased intracellular GSH levels at 7, 12, and 24 h after exposure. However, the decrease in GSH levels was more prominent in response to crocidolite asbestos (average decrease of 55%) at all time points compared to the decrease observed with Libby asbestos (average decrease of 76%). Wollastonite did not decrease the intracellular GSH levels compared to time-matched controls. These results indicate that exposure to Libby and crocidolite asbestos induces oxidative stress in murine macrophages.
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Libby Amphibole Does Not Promote the Formation of 8-oxo-dG or Single Stranded DNA Breaks in Macrophages
RAW264.7 cells were exposed to Libby asbestos, crocidolite asbestos, and wollastonite fibers and the relative levels of 8-oxo-dG were determined as previously described (Bolin et al., 2004
) in order to determine whether the increased levels of ROS generated by Libby and crocidolite asbestos result in oxidative DNA damage. The relative level of 8-oxo-dG is quantified as a ratio of 8-oxo-dG compared to deoxyguanosine. Only adherent cells were included in the DNA damage assay. The levels of 8-oxo-dG in macrophages were significantly elevated in response to crocidolite asbestos at 12 and 24 h (p < 0.05) (Fig. 7A). However, in the same cell type, Libby asbestos did not increase the relative levels of 8-oxo-dG at either time 12 or 24 h. Wollastonite, did not increase the relative levels of 8-oxo-dG at either 12 or 24 h. In response to Libby and crocidolite asbestos, the extent of DNA damage, specifically strand breaks, was also quantified at 24 h through the comet assay (Fig. 7B). Crocidolite asbestos generated a significantly higher level of DNA damage as determined by the percent of cells in the sub-G1 phase (p < 0.05). Libby amphibole did not produce a significant difference in the percentage of cells in the sub-G1 phase. These data indicate that Libby asbestos does not induce oxidative DNA damage in murine macrophage cells and suggests Libby asbestos induces separate cellular responses in vitro compared to crocidolite asbestos.
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Effect of Libby Asbestos on Ogg1 Activity
Ogg1 is an important DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-dG lesions from damaged DNA and its activity has been shown to increase during oxidative stress (Sava et al., 2004
). To determine whether the lack of DNA damage with Libby asbestos was a result of increased DNA repair generated by asbestos-induced oxidative stress, the activity of Ogg1 was quantified as previously described (Bolin et al., 2004). Murine macrophages were exposed to fibers for 12 and 24 h and the activity of Ogg1 was quantified as the percent of cleaved synthetic oligonucleotide, which contains an 8-oxo-dG residue, compared to the total amount of oligonucleotide. As seen in Figure 7C, the activity of Ogg1 in macrophages exposed to Libby asbestos and wollastonite fibers was not different at either time point compared to untreated control cells. Exposure to crocidolite asbestos induces a significant increase in Ogg1 activity in murine macrophages after 12 h, however, Ogg1 activity returns to control levels after 24 h. These results indicate that exposure to Libby asbestos does not increase the activity of Ogg1 in murine macrophages. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The community members of Libby, MT, have experienced significant exposure to amphibole asbestos and have developed numerous ARD due to the asbestos-contaminated vermiculite mine near the community (McDonald et al., 2004
; Whitehouse, 2004). The type of amphibole fiber that residents have been exposed to is a distinct type of amphibole asbestos, which is composed of several different fiber types (Meeker et al., 2003). Because this asbestos type has not been previously studied, we determined the cellular effects of these fibers in order to elucidate a possible cellular mechanism for the initiation of ARD. Our study utilized a phagocytic cell line that is characteristic of alveolar macrophages as well as primary alveolar macrophages that interact and clear inhaled particles in the lung (Xia et al., 2006).
Our data indicate that macrophages internalize Libby asbestos fibers and that the fibers localize to the cytosol and to cytoplasmic vacuoles that frequently surround the nucleus. These data are in accordance with previous results that establish crocidolite fibers are internalized through a microtubule dependent mechanism and localize near the nucleus (Cole et al., 1991). The majority of internalized Libby asbestos fibers are 2 µm or less in length. Libby asbestos fibers of this size have been shown to accumulate in the bark of trees throughout the mine site and surrounding area, indicating that these size fibers are respirable and persist in the environment (Ward et al., 2006). These short, thin fibers are also the predominant asbestos fibers that remain in the tissue of asbestos-exposed humans with pleural plaques and malignant mesothelioma (Suzuki et al., 2005). Hence, these size fibers play an important role in the development of fibrosis and lung cancer (Dodson et al., 2003).
Exposure to Libby asbestos increases the level of intracellular ROS in murine macrophages and can be significantly reduced by exogenous PEG–SOD. These results suggest that the superoxide anion contributes to ROS generated by Libby asbestos exposure. To support the argument that superoxide contributes to asbestos-induced ROS, Libby asbestos exposure also increases DHE fluorescence. DHE is known to preferentially detect superoxide (Zhao et al., 2003). Together these data indicate that Libby asbestos exposure increases intracellular ROS levels in macrophages and that the major contributing ROS is the superoxide anion.
We hypothesized that the increase in superoxide may be due to an alteration of antioxidant concentrations within exposed cells. Therefore, intracellular SOD activity was quantified with an assay that measures total SOD activity and cannot differentiate between the activity of intracellular copper–zinc SOD and mitochondrial manganese SOD. Exposure to Libby asbestos causes a significant decrease in total SOD activity after 3 h of exposure. The decrease in total SOD activity would consequently generate the observed increase in superoxide levels due to Libby asbestos exposure. The reduction in SOD activity by Libby asbestos may be due to posttranslational modifications of SOD as a result of fiber internalization (Marks-Konczalik et al., 1998). In contrast, crocidolite asbestos increases total SOD activity after 24 h, which has been previously reported in mesothelial cells (Cardinali et al., 2006). Chronic exposure to crocidolite asbestos has also been shown to increase SOD activity in vivo and in vitro (Janssen et al., 1992; Mossman et al., 1986). SOD is highly expressed in cells within the lung, such as alveolar macrophages (Kinnula and Crapo, 2003). The increased SOD activity observed during amphibole exposure may be a protective antioxidant response in response to the increased levels of free radicals generated by amphibole internalization.
Exposure to Libby asbestos results in an increase in ROS in conjunction with a significant decrease in intracellular GSH. The decrease in GSH with Libby asbestos was most notable after 7 h and remained significantly decreased after 24 h of exposure. Crocidolite also significantly decreased GSH levels after 7 h of exposure. These data are in accordance with previously published results that indicate crocidolite asbestos exposure results in a significant decrease in intracellular GSH level in both mesothelial and epithelial cells (Golladay et al., 1997; Janssen et al., 1995). In the present study, crocidolite asbestos induces a greater decrease in GSH levels than Libby asbestos, which coupled with the increased cytotoxicity of this fiber, correlates to a higher level of oxidative stress in RAW264.7 cells (Xiao et al., 2003).
Unlike previous studies that utilize only a single amphibole fiber, this study utilizes a sample of Libby asbestos that is a mixture of several amphiboles as well as other fibers not classified in the amphibole family (Meeker et al., 2003). Although exposure to Libby asbestos generates increased levels of ROS, no DNA damage was observed. In contrast, crocidolite asbestos exposure significantly increases DNA damage, which supports previous reports (Fung et al., 1997; Kim et al., 2001). The lack of DNA damage observed with Libby asbestos may be a result of increased DNA repair activity in cells exposed to Libby asbestos. The main defense against oxidative DNA damage is the base excision repair pathway, which is initiated by Ogg1. The activity of Ogg1 significantly increases in murine macrophages in response to crocidolite asbestos, which supports previous in vitro results in alveolar epithelial cells (Kim et al., 2001). However, no difference in Ogg1 activity was observed after exposure to Libby asbestos. Therefore, the lack of DNA damage in macrophages in response to Libby asbestos cannot be explained by increased DNA repair activity in these cells.
Although crocidolite and Libby asbestos are both categorized as amphibole fibers, they are chemically distinct. The major difference between these two fibers is that crocidolite contains a high iron content that is greater than 20% (Kamp and Weitzman, 1999). The high iron content is known to play a role in crocidolite induced DNA damage (Mossman and Gee, 1997; Xu et al., 2002). On the contrary, Libby amphibole contains less than 5% iron content by weight (Meeker et al., 2003). In addition to differences in chemical composition, this study describes separate cellular consequences of each fiber in the same cell line. DNA damage observed with crocidolite asbestos may be an indirect result of the increased SOD activity observed after 24 h (Fig. 8). Increased SOD activity produces excess hydrogen peroxide from the dismutation of superoxide. Increased levels of hydrogen peroxide in crocidolite exposed macrophages may interact with the iron associated with crocidolite asbestos fibers and promote the formation of the hydroxyl radical. Indeed crocidolite fibers have been shown to promote the formation of the hydroxyl radical in the presence of hydrogen peroxide (Kamp and Weitzman, 1999; Kamp et al., 1992). The hydroxyl radical is the most oxidizing ROS in biological systems and promotes the oxidation of 2-dG in DNA generating 8-oxo-dG, which is observed in macrophages in response to crocidolite asbestos (Fig. 7A) (Buettner, 1993). In contrast, Libby asbestos suppresses total SOD activity in macrophages and generates the production of superoxide in exposed cells. The observation that Libby asbestos does not induce DNA damage, may be due to the fact that superoxide has a considerably lower reduction potential and does not participate in the oxidation of 2-dG. Therefore, we hypothesize that the differences in DNA damage observed among Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure (Fig. 8).
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In summary, murine macrophages phagocytize Libby amphibole asbestos fibers, which localize to the cytoplasm and cytoplasmic vacuoles. Internalization of Libby asbestos results in an increase in ROS levels, which can be attributed to the suppression of total SOD activity observed subsequent to Libby asbestos exposure. These results are in contrast to what is observed with crocidolite asbestos, which induces oxidative DNA damage. These data support the premise that the cellular effects observed with different asbestos fibers are mediated by their chemical compositions and the activation of separate cellular mechanisms. Finally, this is the first study to characterize the cellular effects Libby amphibole asbestos, a pathogenic fiber known to cause ARD in humans.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institutes of Health (NIH P20 NCRR017670 and R21 ES012956).
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ACKNOWLEDGMENTS |
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The authors thank Ray Hamilton and Sandra Wells, Center for Environmental Health Sciences (CEHS), University of Montana and James S. Webber, Wadsworth Center, NY, for helpful discussions, Pamela Shaw in the CEHS Fluorescence Cytometry Core for assistance with the Fluorescence activated cell sorting analysis and Anna Marie Ristich, Microscopy Section Manager, at DataChem Laboratories for assistance with the fiber analysis.
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