DNA Double-Strand Breaks by Asbestos, Silica, and ... - ATS Journals

DNA Double-Strand Breaks by Asbestos, Silica, and Titanium Dioxide Possible Biomarker of Carcinogenic Potential? Zola Msiska, Maricica Pacurari1, Anurag Mishra1, Stephen S. Leonard1, Vince Castranova1, and Val Vallyathan 1

Pathology and Physiology Research Branch, Health Effects Laboratory Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia

DNA double-strand breaks (DSBs) can result in cell death or genetic alterations when cells are subjected to radiation, exposure to toxins, or other environmental stresses. A complex DNA-damage–response pathway is activated to repair the damage, and the inability to repair these breaks can lead to carcinogenesis. One of the earliest responses to DNA DSBs is the phosphorylation of a histone, H2AX, at serine 139 (g-H2AX), which can be detected by a fluorescent antibody. A study was undertaken to compare the induction of DNA DSBs in normal (small airway epithelial) cells and cancer cells (A549) after exposure to asbestos (crocidolite), a proven carcinogen, silica, a suspected carcinogen, and titanium dioxide (TiO2), an inert particle recently reported to be carcinogenic in animals. The results indicate that crocidolite induced greater DNA DSBs than silica and TiO2, regardless of cell type. DNA DSBs caused by crocidolite were higher in normal cells than in cancer cells. Silica and TiO2 induced higher DNA DSBs in cancer cells than in normal cells. The production of reactive oxygen species was found to be highest in cells exposed to crocidolite, followed, in potency, by silica and TiO2. The generation of reactive oxygen species was higher in normal cells than in cancer cells. Cell viability assay indicated that crocidolite caused the greatest cytotoxicity in both cell types. Apoptosis, measured by caspase 3/7 and poly (ADP-Ribose) polymerase activation, was highest in crocidolite-exposed cells, followed by TiO2 and silica. The results of this study indicate that crocidolite has a greater carcinogenic potential than silica and TiO2, judged by its ability to cause sustained genomic instability in normal lung cells. Keywords: asbestos; carcinogenesis; DNA damage; H2AX; silica

According to the International Agency for Research on Cancer, asbestos and crystalline silica (silica) are established human lung carcinogens (1–3). Asbestos was first suspected as a carcinogen in 1935, and since then has been well established as a potent pleuropulmonary carcinogen and fibroproliferative agent (4, 5). Although certain epidemiologic and single animal species–specific studies have shown that silica is a carcinogen, there is much scientific disagreement on whether silica causes lung cancer or is only a ‘‘probable carcinogen’’ (6). Unlike asbestos and silica, titanium dioxide (TiO2), has been reported as a nontoxic, nongenotoxic, noncarcinogenic dust, and is widely used in toxicologic and animal exposure studies as a negative control (7–9).

(Received in original form February 16, 2009 and in final form September 17, 2009) This work was supported in part by the U.S. Army Medical Research and Material Command Military Operational Medicine Research Program. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the National Institute for Occupational Safety Health. Correspondence and requests for reprints should be addressed to Val Vallyathan, Ph.D., NIOSH/CDC, 1095 Willowdale Road, Morgantown, WV 26505. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 43. pp 210–219, 2010 Originally Published in Press as DOI: 10.1165/rcmb.2009-0062OC on September 25, 2009 Internet address: www.atsjournals.org

CLINICAL RELEVANCE The results of this study may be valuable in the early detection of exposure to carcinogens in a clinical setting. One of the earliest responses to carcinogens is the doublestranded DNA breaks, resulting in phosphorylation of a histone H2AX. These can be detected in blood of individuals with suspected exposure to carcinogens.

Crocidolite and silica are reported to generate reactive oxygen species (ROS), induce DNA damage, and activate signaling pathways, leading to altered gene expression (10–12). These events are considered important in carcinogenesis. Only ultrafine or nano-size TiO2 is reported to induce significant levels of ROS and induce DNA damage (13, 14). High doses of fine TiO2 have been shown to induce inflammation and tumor formation in rats (15). The inflammation and tumorigenicity induced in the rat model by fine TiO2 is difficult to extrapolate due to the potential excessive particle exposure (overload phenomenon) (16). Nevertheless, the International Agency for Research on Cancer recently classified TiO2 as a Group 2B carcinogen, although epidemiologic studies provided little evidence of carcinogenicity (17). Several studies have shown that the intrinsic ability of fibrous and nonfibrous particulate minerals to induce DNA damage through the generation of ROS can signal cascades of events, leading to phosphorylation of mitogen-activated protein kinases, activation of transcription factors, and expression of early response genes involved in cell proliferation, apoptosis, and carcinogenesis (12, 18–21). As a result of DNA doublestrand breaks (DSBs) induced by particle-generated ROS, alterations in chromatin structure and rapid repair at the damaged sites are initiated by the phosphorylation of H2AX (22). Although active DNA damage repair is initiated in normal cells, enhanced generation of oxidants can induce sustained, irreparable DNA damage, which can initiate cell-cycle arrest and/or transcriptional and post-transcriptional activation of genes associated with repair and apoptosis. These cascades of events and the inability to mitigate DNA repair can lead to genetic instability, clastogenic effects, and development of cancer (23). DNA DSBs are less common compared with single-strand breaks resulting in base damage and other changes in DNA. However, DNA DSBs are considered biologically more important than other types of DNA damage, because their repair is more difficult and, thus, can lead to tumorigenesis if inappropriate resolution of the DSBs occurs. As little as one DNA DSB is sufficient to kill a cell if it inactivates an essential gene or induces apoptosis (24). DNA DSBs are associated with genome instability and predisposition to cancer development (25). Experimental evidence supporting the causal link between DNA DSBs and the induction of gene mutations, chromosomal

Msiska, Pacurari, Mishra, et al.: Predictive Markers of Carcinogenesis

aberrations, and cell transformation that can lead to cancer has also been reported (21). Interactions between the ends of different DNA DSBs can produce translocations in chromosomes, leading to development of carcinogenic products (26). Consequently, detection of persistent, irreparable DNA DSBs may provide an early comparative response measure for agents, such as crocidolite, silica, and TiO2. One of the first steps in the cellular response to DNA DSBs is the phosphorylation of serine 139 at the carboxy terminus of histone H2AX (27). The phosphorylated H2AX, named g-H2AX, appears as discrete nuclear foci at the sites containing DNA DSBs shortly after exposure to exogenous genotoxic agents (28). Each focus represents a single DSB, and is considered a discrete finger-print for denoting the incidence of a DNA DSB that is not repaired (29). H2AX phosphorylation is also an early event in chromosome modification that is followed by apoptotic DNA fragmentation, which constitutes an important step in the course of mammalian apoptosis (30). The rapidly phosphorylated H2AX histone promotes the repair of DNA DSBs to preserve genomic integrity. The aim of this study was to compare the induction of DNA DSBs as a marker of genetic inability to repair DNA damage in normal human small airway epithelial (SAE) cells, and lung cancer cells (A549) exposed to crocidolite (a proven carcinogen), silica (a suspected carcinogen), and TiO2, (low-toxicity particle, recently reported to be a carcinogen). The comparison of the normal human peripheral lung cells with lung cancer cells, containing compromised DNA repair machinery, was considered important to ascertain the validity of this technique as a potential tool to monitor genomic instability and cancer development. Preliminary results of these studies have been previously reported in the form of an abstract (21).

MATERIALS AND METHODS Particles Crocidolite was originally obtained from the Kalahari Desert in South Africa by the National Institute of Environmental Health Sciences at Research Triangle Park, North Carolina. Particle size dimensions were measured by electron microscopy and/or scanning electron microscopy. Surface area measurements were made by Brunauer Emmett Teller (BET) nitrogen adsorption technique and equivalent surface area was calculated from repeated measurements with pure nitrogen, and verified by certified standards. Aerodynamic size measurements and particle elemental analysis were made in 1,000 or more particles by automated image analysis and X-ray spectrometric analysis. The crocidolite fibers had a mean length of 10 mm, mean diameter of 0.21, and surface area of 9.8 m2/g. Crystalline silica (silica [Min-U-Sil]) was obtained from U.S. Silica (Berkeley Springs, WV), and micronized to less than 5 mm with an Accucut Particle Classifier (Donaldson Majal Division, St. Paul, MN). Mass median aerodynamic diameter analysis and automated elemental X-ray analysis of 1,000 or more particles revealed a mean mass median diameter of 3.5 mm and quartz purity of 98.7%, respectively. In addition, the quartz purity was verified by X-ray diffraction spectrometry. Silica particles had a mean surface area of 4.7 m2/g. TiO2 fine, obtained from Aldrich Chemical Co. (Milwaukee, WI), was less than 5 mm in diameter, with a mean surface area of 2.28 m2/g.

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streptomycin. The cells were cultured at 378C in a humidified incubator with 5% CO2.

Exposure to Particles Stock solutions of 200 mg/ml of particles were prepared in RPMI 1640 basal media. All treatments with particles for both cell types were performed in RPMI 1640 basal media containing 0.1% FBS. Working concentrations (100 mg/ml) of the particles were prepared immediately before treatment of the cells in medium containing 0.1% FBSl and vortexed for 2 minutes before use. Sham exposed cells with medium containing 0.1% FBS were used as a negative control.

Lactate Dehydrogenase Assay Lactate dehydrogenase (LDH) activity, as a measure of cell injury and toxicity, was determined in extracellular medium after exposure of cells to particles. Cells (250 3 103) were cultured in six-well plates and allowed to attach for 48 hours before treatment with particles. Cells were then treated with 100 mg/ml of particles for 6, 18, and 24 hours, after which cell suspensions were centrifuged and the supernatant collected for measurement of LDH activity. LDH activity in the supernatant was determined by measuring the reduction of sodium pyruvate in the presence of reduced nicotinamide adenine dinucleotide (Cobas Mira Plus; Roche Diagnostics Systems, Monclair, NJ). LDH activity was expressed as units per liter of cell supernatant. Each experiment was performed three times in triplicate.

Quantification of ROS ROS production was measured by monitoring the fluorescence of the dyes, dichlorofluorescein diacetate (H2DCFDA) and dihydroethidine (DHE) (Molecular Probes, Eugene, OR). The cell-permeable, nonfluorescent H2DCF-DA crosses the cell membrane and accumulates in the cytoplasm, where it is deacetylated by esterases to dichlorofluorescein (DCFH). Oxidation of DCFH by Fenton reaction–generated hydroxyl radicals (OH), H2O2, horseradish peroxidase (HRP), Fe(II), or peroxynitrite results in a nonspecific, highly fluorescent DCF (31– 33). DCFDA for H2O2 is also oxidized to a fluorescent product in the presence of O22 and reactive species, and localizes mainly in the mitochondria (34). Dihydroethydium (DHE), on the other hand, is more specific for superoxide. Cells (1 3 104) were plated in blackwalled, 96-well cell culture plates and allowed to attach for 48 hours. Subsequently, cells were washed twice with PBS and then preincubated with 10 mM H2DCFDA or 5 mM DHE for 30 minutes, and then exposed to 100 mg/ml particles for 2 hours. At the termination of exposure, fluorescence of DCF and DHE was measured with a spectrofluorimeter set at 485-nm excitation and 530-nm emission for H2DCFDA quantification, and 518-nm excitation and 605-nm emission for DHE quantification, respectively (FLx800 Microplate Fluorescence Reader; Bio-Tek Instruments, Winooski, VT). Cells without dye were used to subtract background fluorescence. Each experiment was performed three times in triplicate with a total of nine wells for each particle, giving a total of 27 data points for each particle. In situ ROS generation in cells was also monitored with fluorescence confocal microscopy (Zeiss LSM 510 Axioplan 2; Carl Zeiss Inc., Thornwood, NY). Cells were grown to 70% confluence in chamber slides and then exposed to 100 mg/ml particles for 2 hours. During the last 30 minutes of treatment, 10 mM DCFDA or 5 mM DHE was added. The cells were counterstained with 1 mg/ml 49,6-diamidino-2phenylindole (Molecular Probes, Eugene, OR) for 10 minutes and examined under a confocal microscope set to detect fluorescent DCFH or DHE.

Electron Spin Resonance Assay Cell Cultures Normal human SAE cells (Lonza, Rockland, ME) were grown in small airway epithelial basal medium (SABM) media containing 1% FBS and supplemented with BSA, bovine pituitary extract, insulin, hydrocortisone, gentamicin sulfate amphotericin-B, retinoic acid, transferrin, T3, epinephrine, human recombinant epidermal growth factor (supplied as a bullet kit), and 1% penicillin/streptomycin. The human bronchial epithelial cancer (A549) cell line, from American Tissue Type Culture Collection (Manassas, VA) was grown in RPMI 1640 media containing 5% FBS and 1% penicillin/

The ROS monitoring by the use fluorescent dyes, H2DCF-DA and DHE, are subject to potential artifacts. As such, we investigated the generation of ROS by the conventional, well accepted protocol of electron spin resonance (ESR) spectroscopy with an EMX 300 spectrometer (Bruker Instruments, Billerica, MA), as previously reported (35). A spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was charcoal purified and distilled to remove all ESR-detectable impurities before use. The phosphate buffer was treated with Chelex 100 to remove transition metal ion contamination. Reactants with or without particulate dusts were mixed in test tubes in a final volume of 1.0 ml

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containing 200 mM DMPO, PBS, and 106 A549 cells, and incubated at 378C for 10 minutes with occasional gentle mixing. After the incubation, the reaction mixture was spun for 1 minute and supernatant transferred to a flat cell for ESR measurements. The ESR receiver gain, time constant, sweep time, sweep width, modulation frequency, modulation amplitude, and microwave power were set constant to allow relative intensity comparisons of spectra generated with equal mass of dusts. All measurements were performed at room temperature and under ambient air.

Quantification of DNA DSBs DNA DSBs were determined with a chemiluminescence assay kit for H2AX phosphorylation (Millipore, Billerica, MA), according to the manufacturer’s protocol. The H2AX phosphorylation detection assay kit is a cell-based ELISA for chemiluminescent detection of relative levels of phosphorylated H2AX in microplate cell cultures (Millipore). Briefly, 1 3 104 cells were cultured in 96-well microplates and allowed to attach for 48 hours. The cells in the medium containing 0.1% FBS were then exposed to particles for 24 hours and fixed, followed by permeabilization. Histone H2AX phosphorylated at serine 139 was detected by anti–phospho-H2AX (Ser139), clone JBW301, and an anti–mouse HRP conjugate. The chemiluminescent HRP substrate, LumiGLO, was then added, and signal was measured in a microplate luminometer (EG&G Berthold Microplate Luminometer LB 96V; Postfach, Germany). Each experiment was performed with a control in which cells were labeled with secondary antibody only to estimate the extent of nonspecific binding to the cells. Cells stained with only the secondary antibody were used to subtract substrate-nonspecific chemiluminescence. Each experiment was performed three times in triplicate.

Immunocytochemical Detection of Phosphorylated Histone H2AX Four-chambered slides (Nunc Lab-Tek II; VWR International, West Chester, PA) were seeded with 5 3 104 cells per chamber and allowed to attach for 48 hours before exposure to particles. The cells were at approximately 70% confluence at the time of exposure to the particles. Cells were treated with 100 mg/ml particles in RPMI 1640 basal media for 24 hours. After the treatment, the medium was aspirated and the slides were washed twice with PBS and then fixed with freshly prepared 4% paraformaldehyde by gentle rocking of the slides at room temperature for 10 minutes. Cells were rinsed twice in PBS and made permeable by incubation in 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in PBS for 5 minutes at room temperature. Nonspecific antibody binding was blocked by incubation in 1% (wt/vol) BSA (Sigma Chemical Co.) in TBS for 30 minutes. The cells were then incubated in 250-ml volume of 0.1% BSA containing 2 mg/ml of anti– phospho-H2AX (Ser139), clone JBW301, mouse monoclonal antibody (Millipore). After overnight incubation at 48C, the slides were washed twice with PBS, and then incubated in 250 ml of 1:400 dilution of FITCconjugated goat anti-mouse IgG (Millipore) for 90 minutes at room temperature in the dark. The cells were then counterstained with 1 mg/ml 49,6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) for 10 minutes before viewing under confocal microscope (Zeiss LSM 510 Axioplan 2). g-H2AX foci were counted by eye during the microscopic examination. A total of 40 cells were randomly counted for each treatment with a 1003 objective.

Apoptosis Measured by Caspase 3/7 Activity Caspase 3/7 activity was measured with an Apo-One Homogeneous Caspase 3/7 assay kit (Promega, Madison, WI), according to the manufacturer’s protocol. Briefly, 1 3 104 cells were cultured in 96-well plates and allowed to attach for 48 hours before treatment with particles. Cells were then exposed to100 mg/ml of particles for 3 hours, after which the caspase-Glo 3/7 reagent was added. Addition of the caspase 3/7 reagent resulted in cell lysis, followed by cleavage of the profluorescent substrate, Z-DEVD-R110. Upon cleavage, the substrate produced a green fluorescence, which directly corresponds to the amount of active caspases present in the cell. The fluorescent signal was quantified with an FLx800 Microplate Fluorescence Reader (485 excitation/528 emission; Bio-Tek instruments, VT). The caspase 3/7– protease activity was calculated as fold increase of the fluorescence

signal produced by control cells in medium without particles. Each experiment was performed in triplicates.

Western Blot Analysis of Poly (ADP-Ribose) Polymerase-1 Cells (250 3 103) were grown in six-well plates and allowed to attach for 48 hours. Cells were then treated with 100 mg/ml of particles for 6, 18, and 24 hours. After exposure, cells were washed once with ice-cold 13 PBS and then lysed on ice for 10 minutes with 13 SDS buffer containing 1 ml/ml protease inhibitor cocktail in 1 mM PMSF on ice for 10 minutes (Sigma Chemical Co.). Cell debris was removed by centrifugation for 15 minutes at 14,000 rpm, and 20 mg of protein sample per lane was separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunoblotting for cleaved poly (ADP-Ribose) polymerase (PARP), full PARP, and b-actin was determined with the same membrane after stripping of the blot.

Data Analysis and Statistical Evaluation All data are expressed as means (6SD) of minimum number of three independent experiments performed in triplicates. Comparisons between groups were performed by one-way ANOVA. A P value of less than 0.05 was considered statistically significant.

RESULTS Cytotoxicity of Crocidolite, Silica, and TiO2

Cytotoxicity and damage to the cell membrane integrity, as monitored by the leakage of LDH caused by the exposure of cells to particles, showed that crocidolite was the most toxic particle compared with silica or TiO2 in both cell types (Figures 1A and 1B). In lung cancer A549 cells, only crocidolite induced a substantial enzyme release after 24 hours, which was highly variable and not statistically significant (Figure 1A). Exposure to all three particles demonstrated a time-dependent increase in LDH enzyme activity in normal SAE cells, with crocidolite, silica, and TiO2 causing a greater levels of enzyme release after 18 and 24 hours (Figure 1B). These results demonstrate that normal SAE cells were more susceptible to toxicity induced by all three particles, as evident from the increasing timedependent membrane damage and leakage of LDH. Crocidolite, Silica, and TiO2 Induce the Production of ROS

The ability of crocidolite, silica, or TiO2 to induce the generation of ROS was assessed with H2DCF-DA and DHE. ROS production, as determined by both dyes, was highest in crocidolite-exposed cells, regardless of the cell type, followed in potency by silica and then TiO2 (Figures 2A and 3B). Crocidolite and silica treatments produced significantly higher levels of ROS production in comparison with TiO2 and the untreated control. The production of H2O2 by silica was significantly higher in A549 lung cancer cells than in normal lung SAE cells. The A549 and SAE cells had similar H2O2 production after exposure to crocidolite or TiO2. However, similar levels of the superoxide comparable to H2O2 production were detected in the A549 and SAE cells exposed to all three particulates (Figure 3A). To further confirm by ELISA the ROS generation associated with exposure to these three particulates, cells were treated with fluorescent dyes and particles, and the fluorescent by-products of H2O2 and O22 were monitored in situ by confocal microscopy (Figures 2B, 2C, 3B, and 3C). In the presence of particle exposures, fluorescent signals of both H2O2 and O22 were increased in intracellular organelles. H2O2 fluorescence signal was significantly greater in crocidolite-exposed cells (Figures 2B and 2C). Crocidolite and silica exhibited a moderately similar fluorescence intensity for O22, although the fluorescence signal produced by silica was somewhat weaker (Figures 3B and 3C). These confocal images with H2DCF-DA and DHE for the


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H2O2 and O22 results corroborated the quantitative ELISA measurements (Figures 2A and 3A). To substantiate the generation of reactive species, we used ESR spin trapping of OH radicals generated as a result of the interaction of particles with A549 cells and the scavenging of ROS by catalase and superoxide dismutase (SOD). The ESR technique, with the use of a diamagnetic compound (spin trap) by the addition type reaction of short-lived radicals, will produce a relatively long-lived radical adduct, which can be measured and semiquantitated by ESR. This method is specific and sensitive, and considered to be the preferred technique for the detection and identification of free radical generation. The spectrums displayed show the typical signal intensities generated by the A549 cells containing reagents, and reagents with TiO2, silica, or crocidolite (Figure 4A). The hyperfine 1:2:2:1 quartet splitting is indicative of a DMPO-OH adduct. Preincubation with catalase drastically decreased the signal intensity (75%), and SOD also decreased the signal intensity to a similar degree in all particulate exposures (data not shown). In the case of crocidolite, the scavenging of OH by catalase and SOD were almost identical: approximately 3.2-fold (Figure 4B). Therefore, these results suggest that hydrogen peroxide and super oxide are equally involved in the generation of OH radicals. g-H2AX Detection and Quantitation in A549 and SAE Cells

A quantitative assay for g-H2AX was conducted by ELISA chemiluminescence detection of phosphorylated histone H2AX foci. Exposure to crocidolite induced the greatest levels of g-H2AX, regardless of cell type, followed in potency by silica and then TiO2 (Figure 5A). However, g-H2AX levels were higher in normal SAE cells than in A549 lung cancer cells as compared with control cells. In contrast, g-H2AX levels were greater in A549 lung cancer cells as compared with normal SAE cells after exposure to silica and TiO2. Manual optical counting of g-H2AX foci within cells indicated a different trend from the chemiluminescence results in that silica and TiO2 induced significantly greater numbers of g-H2AX foci in normal SAE cells (Figure 5B). Counting of foci in A549 lung cancer cells corroborated the results obtained with the chemiluminescence assay. In general, the number of foci for all three particulate treatments was greater in cancer A549 cells than in normal SAE cells, including the untreated control cells. These data are supported by immunocytochemical studies. Whereas the induction of DNA DSBs was significant at all time intervals tested in both cell types (data not shown), the responses after 24 hours of exposure were most striking. Exposure to crocidolite induced the greatest g-H2AX foci regardless of cell type, followed in potency by silica and then TiO2 (Figures 5C and 5D). Caspase 3/7 Activation by Crocidolite, Silica, or TiO2

Apoptosis in cell cultures treated with the particles was determined by monitoring caspase 3/7 activity. Caspase 3/7 activity was significantly higher in cells exposed to crocidolite and TiO2 for the normal SAE cells, and not for the A549 lung cancer cells (Figure 6). In addition, an increase in caspase 3/7 activity was obtained for the A549 lung cancer cells at 48 and 72 hours (data not shown). A significant increase in caspase activity was observed with silica or TiO2 exposure as compared with the untreated control cells for both cell types. Effects of Crocidolite, Silica, or TiO2 on PARP

PARP is a nuclear enzyme involved in many cellular processes, including DNA repair, activation of caspases, and apoptosis. In the absence of PARP activation, DNA DSBs will remain unrepaired, leading to clastogenic effects in chromatin. There-

Figure 1. Cytotoxicity assessed by monitoring lactate dehydrogenase (LDH) activity released from (A) A549 lung cancer cells and (B) normal lung small airway epithelial (SAE) cells after exposure to 100 mg /ml crocidolite, silica, or titanium dioxide (TiO2). Data are shown as means (6SD) (n 5 3). *Significant difference from control at P , 0.05.

fore, we investigated the effects of exposure to these particulates in the normal lung and cancer cell lines. Results of these studies show a time-dependent activation of cleaved PARP in cancer cells (Figure 7A). In lung cancer cells, PARP activation by crocidolite occurred at 6 hours, and increased significantly at 18 and 24 hours. Silica activated PARP moderately at 18 and 24 hours, and TiO2 showed a very mild activation only after 24 hours. In the lung cancer cells, crocidolite caused a significant activation of PARP at 18 and 24 hours, and silica showed a moderate activation only after 24 hours (Figure 7A). In the normal SAE cells, only crocidolite induced a mild activation of PARP after 18 and 24 hours (Figure 7B). TiO2 in lung cancer cells and normal SAE cells failed to cause any detectable changes in the activation of PARP, even after 24 hours.

DISCUSSION DNA DSBs are regarded as the most damaging and genotoxic type of DNA damage. Inefficient or inaccurate repair of DNA DSBs can elevate the frequencies of gene translocations, rearrangements, amplification, and deletions, leading to chromosomal instability and neoplastic transformation (23). Very few studies on DNA DSBs due to exposure to crocidolite, silica and TiO2 have been conducted. Marczynski and colleagues (36) reported higher incidences of DNA DSBs in white blood cells of occupationally exposed asbestos workers as compared with the nonexposed control population. Okayasu and colleagues (20) reported enhanced induction of DNA DSBs

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Figure 2. (A) H2O2 generation by A549 and SAE cells pre-exposed to 10 mM of dichlorofluorescein diacetate (H2DCF-DA) for 30 minutes and then exposed to 100 mg/ml of crocidolite, silica, or TiO2 for 2 hours. Values are means (6SD) of three independent experiments in triplicates (n 5 3). *Significant difference from control at P , 0.05. (B) In situ localization of H2O2 generation in A549 control cells (I ), and cells exposed to 100 mg/ml TiO2 (II ), silica (III ), and crocidolite (IV ) for 2 hours in serum-free medium. H2DCF-DA, fluorescing green, was added in the last 30 minutes of treatment. DNA was counterstained with 49,6-diamidino-2-phenylindole (DAPI), fluorescing blue. Note that the bright green fluorescence in cells represents H2O2 generation. (C ) In situ localization of H2O2 generation in SAE control cells (I ), and cells exposed to 100 mg/ml TiO2 (II ), silica (III ), and crocidolite (IV ). Experiments were conducted as described in Figure 2B.

in a DNA repair–deficient cell line, xrs-5 cells, after 24-hour exposure to chrysotile asbestos with contour-clasped homogenous electric field (CHEF) gel electrophoresis. By measuring g-H2AX accumulation, Pietruska and Kane (37) were able to report that SV40 oncoproteins enhance induction of DNA DSBs in murine normal and malignant mesothelial cells ex-

Figure 3. (A) Superoxide generation in A549 and SAE cells preexposed to 5 mM of DHE for 30 minutes and then 100 mg/ml of crocidolite, silica, or TiO2 for 2 hours. Values are means (6SD) of three independent experiments. *Significant difference from control at P , 0.05. (B) In situ generation of O22 in A549 control cells (I ), and cells exposed to 100 mg/ml TiO2 (II ), silica (III ), and crocidolite (IV ) for 2 hours in serum-free medium. DHE fluorescing red was added in the last 30 minutes of treatment. DNA was counter-stained with DAPI, fluorescing blue. The third panel shows merged images of DHE and DAPI staining. (C ) In situ localization of O22 generation in SAE control cells (I ), and cells exposed to 100 mg/ml TiO2 (II ), silica (III ), and crocidolite (IV ). Experiments were conducted as described in Figure 2B.

posed to crocidolite. Accumulation of g-H2AX in MEF cells (transgenic mouse primary embryo fibroblast) exposed to chrysotile asbestos has also been demonstrated (38). In this study, induction of DNA DSBs due to exposure to crocidolite, as measured by accumulation of g-H2AX, was demonstrated in


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Figure 4. Generation of hydroxyl radicals (OH) from A549 cells and media with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), cells exposed to TiO2, cells exposed to silica, and cells exposed to crocidolite. Representative spectra from one experiment are displayed (A). Mean semiquantitative electron spin resonance (ESR) spectra measured from three independent experiments after exposure to particles and 10-minute incubation at 378C under identical conditions (B). All particle exposures were 1 mg/ml in the presence of 200 mM DMPO and phosphate buffer. Catalase (2,000 U/ml) and superoxide dismutase (SOD) (0.5 mg/ml) were used to confirm the OH generation. All ESR parameters were kept similar at a receiver gain of 2.5 3 104; time constant, 0.40 s; modulation amplitude, 1.0 G; scan time, 41 seconds; magnetic field, 3,385 (6100) G.

both normal and malignant human lung cell lines. Although not significantly different, DNA DSBs caused by crocidolite were somewhat higher in the normal cells than the cancer cells. Cells exposed to crocidolite had the greatest number of DNA DSBs in comparison with cells exposed to silica or TiO2, regardless of cell type. The elevated g-H2AX foci in both cell lines suggests either a compromised repair of DNA damage after exposure to crocidolite or, more likely, a continuous production of DNAdamaging effects of this agent on these cells. Silica has been shown to cause DNA DSBs in cell-free systems (39). DNA DSBs due to silica exposure, as measured by accumulation of g-H2AX, has been shown to be reduced in tumor tissues as compared with hyperplastic and advanced preneoplastic tissues of rats, and g-H2AX was not detected at all in the nonexposed normal rat bronchial epithelial cells (40). In contrast, a few H2AX foci were observed in normal as well as cancer cells, even in the absence of exposure to these toxic agents in the present study. Our study has demonstrated a significant increase in g-H2AX in human lung cancer cells

exposed to silica as compared with nonexposed control cells. No significant increase in DNA DSBs in normal cells exposed to silica was observed as compared with the nonexposed control normal cells when using the chemiluminescence kit. However, g-H2AX foci counting indicated a significant increase in DNA DSBs after exposure to silica. The discrepancy in the results can be attributed to potential problems in both methods used to measure DNA DSBs. It is possible that the chemiluminescence kit used is not as sensitive as the manual optical counting of foci. At the same time, the possibility exists that number of foci were overestimated by manual counting. Manual counting of foci by optical microscopy is subjective and variable. Similarly, TiO2 induced a significant amount of DNA DSBs in the cancer cells, and not in the normal cells, as compared with their respective nonexposed control cells when using the chemiluminescence assay. This observation may have significance, because it was shown that ultrafine TiO2 particles, even in the absence of photoactivation, were reported to cause oxidative bronchial epithelial cell DNA damage (7). Manual

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Figure 5. (A) Generation of g-H2AX in A549 and SAE cells exposed to 100 mg/ml crocidolite, silica or TiO2 for 24 hours in serum-free medium. Data were pooled from three independent experiments. Error bars indicate means (6SD) of three independent experiments in triplicates (n 5 3). *Significant difference from control at P , 0.05. (B) Number of g-H2AX foci in A549 and SAE cells exposed to 100 mg/ml crocidolite, silica, or TiO2 for 24 hours in serum-free medium. Data were pooled from three independent experiments. Error bars indicate means (6SD) of three independent experiments in triplicates (n 5 3). *Significant difference from control at P , 0.05. (C ) Intracellular accumulation of g-H2AX foci in control A549 cells (I ), and in cells exposed to 100 mg/ml TiO2 (II ), silica (III ), or crocidolite (IV ) for 24 hours in serum-free medium. The cells were immunocytochemically labeled with phospho-specific g-H2AX antibody, with the secondary antibody fluorescing green. DNA was counter-stained with DAPI, fluorescing blue. (D) Intracellular accumulation of g-H2AX foci in and SAE control cells (I), and in cells exposed to 100 mg/ml TiO2 (II ), silica (III ), or crocidolite (IV ) for 24 hours in serum-free medium. Experiments were conducted as described in Figure 5B.

counting of foci in cells indicated a significant increase in DNA DSBs after exposure to TiO2 in normal SAE cells. The discrepancy in these results can also be attributed to problems in the methods used, as described previously here. This study showed that all three types of particle induced greater levels and numbers of g-H2AX foci in the lung cancer cell line than the normal lung cell line. Given the nature of the aneuploid cancer cell line, A549 (i.e., with chromosomal abnormalities [16, 23]), it can be hypothesized that these cells are more susceptible to genotoxic effects of silica and TiO2 than are the normal cells. It is also possible that the A549 cells, as opposed to the normal cells, repair the initial DNA damage poorly and/or lack induction of protective systems. Clearly, the ability of crocidolite, silica, and TiO2 particles to induce DNA damage differs between A549 and SAE cells.

Histone H2AX is phosphorylated on Ser139, not only in response to DNA damage caused by environmental genotoxic factors, but also in healthy, untreated cells. This has been called ‘‘intrinsic,’’ ‘‘programmed,’’ or ‘‘scheduled’’ H2AX phosphorylation (29, 41). In the absence of externally induced DNA damage, phosphorylation of H2AX occurs primarily during DNA replication. The extent of H2AX phosphorylation during the cell cycle varies significantly, depending on cell line (42, 43). In the absence of any treatment, both A549 and SAE cells expressed phosphorylated g-H2AX at a basal level. A greater number of g-H2AX foci were observed in the untreated lung cancer A549 cells than the untreated normal SAE lung cells. This finding is in contrast to the findings of Huang and colleagues (29), who reported a significant expression of g-H2AX in untreated normal human bronchial epithelial cells versus


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Figure 6. Caspase 3/7 activation in A549 and SAE cells after exposure to 100 mg/ml of crocidolite, silica or TiO2 for 24 hours. Values are means (6SD) of three independent experiments. *Significant difference from control at P , 0.05.

untreated cancer cells (A549). The expectation is that the number of H2AX foci should be higher in the aneuploid cancer cell line, A549, containing more chromosomes, and thus a larger area for DNA DSB formation than the normal cell line, with a lower number of chromosomes. DNA damage is a well known stimulus for apoptosis (11). DNA DSBs are formed in the course of DNA fragmentation in apoptotic cells (29). An increase in caspase 3/7 activity, concomitant with an increase in DNA DSBs caused by crocidolite in both cell types, suggests that the DNA DSBs observed may be the result of both the damaging effects of crocidolite as well as DNA DSBs generated by apoptosis-associated DNA fragmentation. Significant increases of DNA DSBs in crocidolite-exposed cells were seen within 1 hour of treatment, and increased thereafter with longer exposure times. According to Huang and Darzynkewicz (44), g-H2AX DNA DSBs induced by external DNA-damaging agents are seen early during the treatment (10 min to 2 h), whereas apoptosis-associated g-H2AX DNA DSBs are seen later (.3 h). The data presented in this study are in agreement with several reports whereby crocidolite was shown to cause apoptosis in epithelial cells (45–47). Crocidolite-induced apoptosis may account for the pathogenic effects of the fibers (11). DNA damage induces the enzymatic activation of PARP, and PARP protein is cleaved during apoptosis to signal repair pathways that contribute to posttranslational modification of histones and nuclear proteins. This study showed that exposure to crocidolite, in both cell types, led to a time-dependent, significant activation of cleaved PARP. Activation of cleaved PARP due to crocidolite exposure implies that enhancement of DNA repair is impaired. In contrast, silica showed only a moderate activation of PARP after 24 hours, and TiO2 failed to induce any detectable activation at all the times investigated. Both A549 and SAE cells had a significant increase in caspase activity due to silica exposure, as compared with that of the nonexposed control cells. The implication is that the observed DNA DSBs are due to the damaging effects of silica, in addition to being generated by apoptosis-associated DNA fragmentation. Like crocidolite, activation of cleaved PARP was also observed in both cell types due to silica exposure. This result also implies that enhancement of DNA repair is impaired in cells exposed to silica. Similar to crocidolite, DNA DSBs induced by exposure to TiO2 may be due to the damaging effects of TiO2, in addition to being generated by apoptosisassociated DNA fragmentation. This study has provided considerable insight into the molecular mechanisms underlying asbestos-induced disease development. Crocidolite, a well documented, proven pulmonary carcinogen, induced greater levels of ROS, leading to enhanced

DNA damage and apoptosis, and probably perpetuating cascades of molecular events pivotal in carcinogenesis. Among the many molecular mechanisms investigated, accumulating evidence implicates ROS-induced DNA DSBs as a major event to signal a cascade of events leading to phosphorylation of mitogen-activated protein kinase and activation of transcription factors, promoting the activation of early response genes, leading to cell proliferation and carcinogenesis. The fact that crocidolite, silica, and TiO2 are biopersistent particles may result in the generation of a continued increased level of ROS, although there are significant differences in the extent that these three particulates produce ROS. The data in this study support the hypothesis that ROS may be one of the many mechanisms involved in crocidolite-induced DNA damage and apoptosis, as results show a significant increase in H2O2 and the superoxide produced by both cell types. Similarly, the data in this study suggest that silica and TiO2 induce DNA DSBs via the production of ROS in both cell lines. ROS may also be involved in

Figure 7. (A) Effect of crocidolite, silica or TiO2 exposure on the activation of poly (ADP-Ribose) polymerase (PARP) in A549 lung cancer cells. The cells were treated with 100 mg/ml particles for the indicated time points, and cell lysates were prepared and analyzed for PARP cleavage by Western blot. The blots were probed with b-actin antibody to confirm equal loading of samples. The experiments were performed three times, and a representative blot is shown. (B) Effect of crocidolite, silica, or TiO2 exposure on the activation of PARP in SAE normal lung cells. Experiments were conducted as described in Figure 7A.

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the induction of apoptosis in cells exposed to TiO2, as demonstrated in Syrian hamster embryo fibroblasts (48). In conclusion, this study demonstrates the induction of DNA DSBs by crocidolite in both normal and lung cancer cells. DNA DSBs are a major type of DNA damage that can lead to translocations and chromosomal instability, two important mechanisms in the generation of malignant tumors. Evidence presented in this study and the literature suggests that crocidolite has a greater ability than silica or TiO2 to induce carcinogenesis by causing sustained genomic instability. Detection of these DNA DSBs may provide a measurement of the potential cancer risk in individuals exposed to crocidolite. Author Disclosure: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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