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Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals
Free Radical Biology & Medicine, Vol. 25, No. 6, pp. 728 –739, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00158-0
Original Contribution CIGARETTE SMOKE AUGMENTS ASBESTOS-INDUCED ALVEOLAR EPITHELIAL CELL INJURY: ROLE OF FREE RADICALS DAVID W. KAMP,* MARC J. GREENBERGER,* JANE S. SBALCHIERRO, SCOTT E. PREUSEN,* SIGMUND A. WEITZMAN†
and
Department of Medicine, pDivision of Pulmonary Medicine, and †Hematology–Oncology, Northwestern University Medical School and Veterans Affairs Chicago Health Care System, Lakeside Division, Chicago, IL, USA (Received 16 March 1998; Revised 12 June 1998; Accepted 12 June 1998)
Abstract—Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not established. Alveolar epithelial cell (AEC) injury due to oxidant-induced DNA damage and depletion of glutathione (GSH) and adenosine triphosphate (ATP) may be one important mechanism. We previously showed that amosite asbestosinduces hydroxyl radical production and DNA damage to cultured AEC and that phytic acid, an iron chelator, is protective. We hypothesized that whole cigarette smoke extracts (CSE) augment amosite asbestos-induced AEC injury by generating iron-induced free radicals that damage DNA and reduce cellular GSH and ATP levels. Asbestos or CSE each caused dose-dependent toxicity to AEC (WI-26 and rat alveolar type I-like cells) as assessed by 51chromium release. The combination of asbestos (5 mg/cm2) and CSE (0.01– 0.1%) caused synergistic injury whereas higher doses of each agent primarily had an additive toxic effect. Asbestos (5 mg/cm2) augmented CSE-induced (0.01–1.0%) AEC DNA damage over a 4 h exposure period as assessed by an alkaline unwinding, ethidium bromide fluorometric technique. These effects were synergistic in A549 cells and additive in WI-26 cells. Asbestos (5 mg/cm2) and CSE (0.5–1.0%) reduced A549 and WI-26 cell GSH levels as assessed spectrophotometrically and ATP levels as assessed by luciferin/luciferase chemiluminescence but a synergistic interaction was not detected. Phytic acid (500 mM) and catalase (100 mg/ml) each attenuated A549 cell DNA damage and depletion of ATP caused by asbestos and CSE. However, neither agent attenuated WI-26 cell DNA damage nor the reductions in GSH levels in WI-26 and A549 cells exposed to asbestos and CSE. We conclude that CSE enhance asbestos-induced DNA damage in cultured alveolar epithelial cells. These data provide additional support that asbestos and cigarette smoke are genotoxic to relevant target cells in the lung and that iron-induced free radicals may in part cause these effects. © 1998 Elsevier Science Inc. Keywords—Asbestos, Cigarette smoke, Alveolar epithelial cells, DNA damage, Reactive oxygen species, Lung injury, Glutathione, ATP, Free radical
INTRODUCTION
Alveolar epithelial cell (AEC) injury is one important event that may perpetuate lung damage caused by asbestos and cigarette smoke. Asbestos is internalized by alveolar type I cells resulting in cytotoxicity and triggering of a proliferative response in alveolar type II (ATII) cells [3]. Studies in animals and humans demonstrate that cigarette smoke promotes the pathogenic effects of asbestos by impairing lung clearance of the fibers in part by augmenting asbestos uptake across the pulmonary epithelium [4]. Several lines of evidence strongly implicate reactive oxygen species (ROS) generated by cigarette smoke and asbestos as one important mechanism causing synergistic lung damage. First, cigarette smoke and asbestos each generates significant levels of ROS [5–7]. Cigarette
Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not yet established [1,2]. Although epidemiologic evidence attributes bronchogenic carcinoma to a multiplicative interaction between tobacco smoke and asbestos, a simple additive response has also been reported [1,2]. Despite extensive investigations exploring the interactive effects between cigarette smoke and asbestos, the precise mechanisms involved at the cellular and molecular level are unclear. Address correspondence to: David W. Kamp, M.D., Northwestern University Medical School, Pulmonary Division; Passavant, Room 777, 303 East Superior Street, Chicago, IL 60611, USA; Tel: (312) 908-8163; Fax: (312) 908-4650; E-Mail: [email protected] 728
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smoke is a complex mixture containing over 5000 different substances [8]. Cigarette tar, which contains at least 1017 free radicals per gram [8], is a stable substance that can accumulate iron that promotes the formation of highly reactive hydroxyl radicals (•OH) [8,9]. The gas phase of cigarette smoke also contains short-lived ROS that are potent oxidants [5]. Iron-induced •OH are implicated in causing genotoxicity in cells exposed to aqueous whole cigarette smoke extracts (CSE). Ferrous iron in asbestos in the presence of hydrogen peroxide (H2O2) also produces •OH via a Fenton reaction in the following manner [7]:
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duced AEC injury in vitro by generating iron-catalyzed free radicals that damage DNA and reduce cellular GSH and ATP levels. We demonstrate that CSE increase amosite asbestos-induced cytotoxicity and DNA-SB formation in cultured AEC. In addition, synergistic DNA damage was noted in A549 cells and these effects were attenuated by phytic acid and catalase. Both agents caused AEC DNA damage without necessarily altering GSH and ATP. METHODS
Mineral dusts H2O2 1 Asbestos-Fe21 3 •OH1OH1 Asbestos-Fe31 Jackson and coworkers [10] demonstrated that CSE synergistically augment asbestos-induced •OH formation detected by electron paramagnetic resonance spectroscopy and bacteriophage PM2 DNA strand break (DNASB) formation that were both prevented by •OH scavengers or iron chelators. Second, cigarette smoke-induced fiber uptake by pulmonary epithelial cells is diminished by antioxidant enzymes (AOE) or iron chelators [4]. Third, AOE, such as catalase and superoxide dismutase, or iron chelators each attenuate asbestos-induced AEC injury by reducing lipid peroxidation, DNA-SB formation, and 8-OH-deoxyguanosine base pair changes in DNA [5,7,10 –12]. Exogenous AOE and reduced glutathione (GSH) also diminish cigarette smoke-induced A549 cell DNA damage and altered permeability [13, 14]. Fourth, as recently reviewed [5], ROS generated by inflammatory cells in vitro and in vivo may contribute to pulmonary toxicity. Finally, asbestos and other oxidant stresses alter the expression of AOE in the pulmonary epithelium suggesting important adaptive responses [15]. Although these data firmly support the hypothesis that ROS are one mechanism that cigarette smoke and asbestos synergistically injure the lungs, the effects of these agents on an important target cell, AEC, are unknown. The cellular and molecular events culminating in AEC injury after an oxidant stress from asbestos and cigarette smoke are uncertain. DNA strand breaks are one of the earliest cellular changes occurring after an oxidant stress from H2O2 or radiation [16,17]. ROSinduced DNA damage can subsequently deplete GSH and ATP levels resulting in cell death [5,14,17]. Cigarette smoke or asbestos each alone cause DNA base pair modifications and strand break formation [6,13,18 –21]. We previously showed that asbestos-induced AEC toxicity is due in part to DNA-SB formation caused by the production of iron-induced •OH-like species [11]. We hypothesized that CSE augment amosite asbestos-in-
Amosite asbestos used in these experiments was Union International Contre Le Cancer (UICC) Reference Standard samples kindly supplied by Dr. V. Timbrell [22]. A stock concentration of amosite (5 mg/ml) was prepared in Hanks balanced salt solution plus calcium, magnesium and 15 mM N-2-hydroxyethylpiperazine-N92-ethanesulfonic acid (HBSS). The suspension was autoclaved and stored at 4°C. Before use, the mineral dusts were warmed to 37°C and vigorously vortexed to ensure a uniform suspension. Cigarette smoke extract preparation Cigarette smoke extracts were prepared fresh for each experiment as previously described [10,23]. Briefly, CSE were obtained by bubbling cigarette smoke from 1 commercial unfiltered cigarette (Pall Mall) through phosphate buffered saline (PBS) (10 ml) at 3 puffs/min. It is estimated that the cigarette smoke extract concentration, which simulates the concentration present in adult smoker’s lung, is approximately 0.1% (vol/vol) [23]. As a result, AEC were exposed to various dilutions of CSE (0.01–1.0% vol/vol) for variable periods with and without asbestos present. Because Holden and colleagues [23] showed that CSE containing 1 cigarette/ml (100% vol/vol) have approximately 1 mg/ml of nicotine, we estimated that the final concentration of nicotine in the cultures varied from 0.1–10 mg/ml. Alveolar epithelial cells SV-40 transformed WI-26 cells (VA4; CCL-95.1) and A549 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). WI-26 cells are derived from human embryonal lung. Electron microscopic analysis of WI-26 cells in our laboratory have revealed type-1 pneumocyte-like morphology in that they have tight junctions and no lamellar bodies. WI-26 cells were maintained in Eagles minimal essential medium (GIBCO, Grand Island, NY, USA) with L-
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glutamine (0.3 mg/ml), nonessential amino acids, penicillin (100 U/ml), streptomycin (200 mg/ml), and amphotericin B (0.25 mg/ml), and supplemented with 20% fetal bovine serum (FBS; GIBCO) and grown in 5% CO2 at 37°C. A549 cells, which are human bronchoalveolar carcinoma-derived cells with synthetic and morphologic characteristics of alveolar epithelial type II cells, were maintained in Dulbecco’s modification of Dulbecco’s modified Eagle medium (DMEM) supplemented as above only with 10% FBS. Rat ATII cells were isolated from specific pathogenfree adult Harlan–Sprague–Dawley rats (200 –250 g) as previously described [15,17]. Briefly, the cells were obtained from rat lungs by elastase digestion, followed by mincing and sequential filtration. ATII cell purity was enhanced by “panning” the cell suspension on plates coated with rat immunoglobulin G (IgG 500 mg/ml; Sigma). The cell suspension was then washed, resuspended in DMEM plus 10% FBS with antibiotics, and plated at a density of 5 3 105 cells/well in a 24-well plate (Costar, Cambridge, MA, USA). After isolation, cell viability was .90% by trypan dye exclusion and type II cell purity 24 h after isolation was 80 to 85% ATII as assessed by a modified Papanicolaou stain (n 5 4). The cells were incubated in 95% air/5% CO2 at 37°C for 7 to 10 days before use. Alveolar type II cells cultured for 7 to 10 days (RAEC) gradually lose their characteristic features (i.e., lamellar bodies and surfactant production) and morphologically resemble alveolar type I cells [24]. RAEC were chosen for these experiments because type I cells comprise nearly 95% of the alveolar surface area and asbestos is noted in these cells early after exposure [3]. Previous studies in our laboratory have shown that cell purity assessed by tannic acid staining of lamellar bodies varies with time in culture (% lamellar body staining: 24 h: 86 6 1%; 48 h: 80 6 3%; 5 d: 37 6 2%; and 7–10 d: 6 6 2%; n 5 3–6 at each time point) and that over 95% of the cells contain cytokeratin by indirect immunoflouresence with anticytokeratin antibody after 5 to 7 days in culture [25]. Cytotoxicity assay Cytotoxicity was measured by a 51Cr release assay previously described by our laboratory with some minor modifications [11,25]. Briefly, WI-26 cells were grown to confluency in gelatin-coated 24 well (2 cm2) tissue culture plates, washed twice with sterile HBSS, radiolabeled with 1.0 mCi/well Na251CrO4 (ICN Pharmaceuticals, Irvine, CA, USA) for 18 h in media supplemented with 2% FBS and then washed three times with HBSS to remove unbound 51Cr. The radiolabeled WI-26 cells were then incubated with one of the test conditions in HBSS. In some experiments, catalase or phytic acid
(Sigma) was added prior to the CSE and asbestos. After incubation for 18 h at 37°C in 95% air/5% CO2, the medium bathing the WI-26 cells (1 ml) was aspirated and the WI-26 cells were washed three times with 0.5 ml of HBSS. An aliquot of the combined aspirate and wash (Media) was placed into a test tube and counted in a gamma counter (Beckman Instruments, Palo Alto, CA, USA). The total possible counts (Total) were determined from control cell lysates (1 ml 1N NH4 OH 3 10 min) plus control Media. Injury to the WI-26 cells under each experimental condition was then calculated by the percent of 51Cr release defined as: $~test Media cpm 2 control Media cpm!/total cpm%) 3 100. This cytotoxicity index represents cell lysis and detachment corrected for background spontaneous 51Cr release from control WI-26 cells assayed the same day. Spontaneous 51Cr release from WI-26 cells incubated 18 h under control conditions (HBSS) was 30.5 6 2.6% (n 5 15). Cell number (data not shown), viability ($90% trypan blue dye exclusion), and morphology were similar in WI-26 cells maintained under control conditions compared with cells maintained in media supplemented with FBS. DNA Strand Break (DNA-SB) The formation of DNA-SB were assessed by alkaline unwinding and ethidium bromide fluorescence, as previously described [11]. Because ethidium bromide preferentially binds to double stranded DNA in alkali, the relative amounts of nonbroken double-stranded DNA and broken single-stranded DNA can be assessed [17]. The alveolar epithelial cells (WI-26, A549, and RAEC) were exposed to various concentrations of CSE and asbestos for variable periods. In some experiments, phytic acid or catalase was added prior to the CSE and asbestos. Following incubation with asbestos, the cells were washed three times with HBSS buffer and then analyzed as follows: 100 ml of solution B (0.25 M mesoinositol, 10 mM sodium phosphate, 1 mM MgCl2; pH 7.2) followed without mixing with 100 ml solution C (9 M urea, 10 mM NaOH, 2.5 M cyclohexanediamineacetate and 0.1% sodium dodecyl sulfate) were added to each well and then incubated at 0°C for 10 min to enable cell lysis and chromatin disrupture. Without mixing, 50 ml of solution D (0.45 vol solution C in 0.2 N NaOH) and 50 ml solution E (0.40 vol solution C and 2 N NaOH) were added and incubated at 0°C for 30 min. After incubation, 200 ml of solution F (1M glucose and 14 mM mercaptoethanol) was added to each well followed by
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sonication (Sonifier Cell Disrupter; Heat Systems Ultrasonics, Plainview, NY, USA) for 3 s and addition of 750 ml of solution G (6.7 mg/ml ethidium bromide in 13.3 mM NaOH). Fluorescence was then determined using a Sequoia Turner fluorometer (Model 450; Mountain View, CA, USA) with excitation at 520 nm and emission at 585 nm. The percentage of double stranded DNA ~% ds-DNA) 5 (F 2 Fmin)/(Fmax 2 Fmin) 3 100
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Glutathione assay Total glutathione was determined as previously described [26]. The assay is based on the rate of decrease of 5-59-dinitrobis(-2-nitrobenzoic acid) measured spectrophotometrically at 412 nm. AEC were exposed for 4 h at 37°C to various concentrations of CSE with or without asbestos before GSH levels were determined and expressed per cell number.
ATP Measurement where F is the fluorescence of the experimental condition, Fmin the background ethidium bromide fluorescence determined after converting all the DNA into single strand form, and Fmax is the fluorescence determined after adding the mercaptoethanol solution before the alkaline solution to maintain the pH 11.0, which is well below that needed to augment unwinding of single stranded DNA. Results are expressed as DNA-SB defined as 100 2 % ds-DNA. The amount of single stranded DNA present in alkali after sonication is proportional to the number of DNA-SB (either single or double) because DNA-SB varies directly as a function of the extent of DNA unwinding [10]. We previously showed that the amount of asbestos used in these experiments (10 mg/ml or 5 mg/cm2) does not quench the fluorescent signal nor cause DNA damage while harvesting the cells [11].
Cellular ATP levels were assessed by the luciferin/ luciferase chemiluminescence technique, as previously described [26]. Confluent WI-26 and A549 cells were washed three times, and then exposed to various concentrations of CSE with or without asbestos (10 mg/ml). After incubation for 18 h at 37°C, the cells were washed three times, heated to 95°C for 4 min in 2 ml of KH2PO4 and 4 mM MgPO4 and placed on ice. Within 4 h of sample collection, ATP levels were be assayed by combining 1 ml of sample with 2 ml of 50 mM NaAsO2, 20 mM MgSO4 and 50 ml of luciferin/luciferase. Light emission was quantified precisely 15 s after the addition of luciferin/luciferase. ATP levels were determined from standards prepared in parallel dishes not containing AEC but otherwise handled in a similar manner as described. This assay has been shown to be linear for 1029 to 1025 M ATP. The data are expressed as nM ATP per 106 cells.
Fig. 1. Cigarette smoke extracts (CSE) and amosite asbestos (ASB) injure WI-26 cells. WI-26 cells were exposed to CSE (0.01 to 1.0%) and/or ASB (5 mg/cm2) for 18 h. Cell cytotoxicity was assessed based upon the percentage of 51Cr-released corrected for the spontaneous release from control cells as defined in the Methods section. Asbestos-induced WI-26 cell cytotoxicity was synergistically increased by low-dose CSE (0.01%) and additively increased at higher doses of CSE (0.1–1.0%). Data expressed as the mean 6 SEM (n 5 8). *p , .01 vs. ASB and/or CSE; † p , .05 vs. control; § p , .001 vs. control.
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Statistical Analysis The results of each experimental condition were determined from the mean of duplicate or triplicate trials. Unless otherwise noted, at least 6 separate experiments (n 5 6) were performed for each group of experiments and the data expressed as the mean 6 SEM. A twotailed, unpaired Student’s t-test was used to assess significance between two groups of data. Analysis of variance was used when comparing more than two groups; differences between two groups within the set were analyzed by a Fischer’s protected least significant difference test. A synergistic effect was defined when the observed effect caused by CSE and asbestos was greater than the sum of each agent. p-values , .05 were considered significant. RESULTS
cific 51Cr-release after an 18 h exposure period. CSE resulted in dose-dependent toxicity to WI-26 cells. The combination of CSE (0.1 and 1.0% vol) and asbestos (5 mg/cm2) had an additive cytotoxic effect. However, synergistic WI-26 cells injury occurred with CSE (0.01% vol) and asbestos ( p , .01 vs. asbestos plus CSE). Higher concentrations of asbestos (25–250 mg/cm2) with CSE caused primarily additive toxic effects (data not shown). As shown in Fig. 2A, amosite asbestos (5 mg/ cm2) alone caused modest injury to primary isolated RAEC after an 18 h exposure period (6% Sp 51Crrelease; p , .05). Similar to WI-26 cells, CSE (0.1% vol) plus asbestos (5 mg/cm2) also induced synergistic injury to RAEC (Fig. 2A). Therefore, CSE augments amosite asbestos-induced injury to WI-26 and RAEC cells. CSE augment asbestos-induced AEC DNA Damage
CSE increase asbestos-induced AEC cytotoxicity As shown in Fig. 1, amosite asbestos (5 mg/cm ) alone caused negligible injury to a human pulmonary epithelial-like cell line (WI-26 cells) as assessed by spe2
Fig. 2. (A) Cigarette smoke extracts (CSE) and amosite asbestos (ASB) synergistically injure RAEC cells. RAEC cells were exposed to CSE (0.1%) and/or ASB (5 mg/cm2) for 18 h. Cell cytotoxicity was assessed based upon the percentage of 51Cr-release corrected for the spontaneous release as defined in the Methods section. Data expressed as the mean 6 SEM (n 5 6). (B) Cigarette smoke extracts (CSE) and amosite asbestos (ASB) cause DNA damage in RAEC cells. RAEC cells were exposed to CSE (0.1%) and/or ASB (5 mg/cm2) for 4 h. DNA-SB formation was assessed as defined in the Methods section. Data expressed as the mean 6 SEM (n 5 5). *p , .05 vs. control; † p , .02 vs. ASB alone, CSE alone, or ASB plus CSE.
We determined whether CSE and asbestos induce AEC DNA damage. As shown in Fig. 3, CSE alone caused dose-dependent WI-26 cell DNA-SB formation after a 4 h exposure period (r 5 0.94; p , .05). In contrast to these type I-like cells, CSE induced minimal levels of DNA-SB formation in A549 cells, a cell line possessing type II cell features. Significant A549 cell DNA damage was noted only after exposure to the highest dose of CSE tested (Fig. 3). Therefore, CSE cause DNA damage to AEC and these effects are cell typedependent. As illustrated in Fig. 4, CSE (0.01–1.0%) and asbestos (5 mg/cm2) each alone induced DNA-SB formation in
Fig. 3. DNA strand breaks induced by CSE in WI-26 and A549 cells. WI-26 and A549 cells were exposed to CSE (0 –1.0%) for 4 h and then the percentage of DNA-SBs were determined by the alkaline unwinding, ethidium bromide fluorometric technique described in the Methods section. CSE caused dose-dependent DNA-SB formation in WI-26 cells (solid bars) and lesser amounts of DNA-SB in A549 cells (stippled bars). Data expressed as the mean 6 SEM (WI-26 cells: n 5 7; A549 cells: n 5 6). *p , .05 vs. control.
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occurred with negligible changes in cell number or viability (data not shown), cytotoxicity was noted by 18 h (Figs. 1 and 2A). We conclude that CSE plus asbestos induce DNA damage in WI-26 cells and RAEC that is less than or comparable to an additive effect of each agent alone but is synergistic in A549 cells. CSE reduce GSH and ATP levels in AEC
Fig. 4. CSE and asbestos cause DNA-SB in WI-26 and A549 cells. WI-26 (top graph) and A549 (bottom graph) cells were exposed to amosite asbestos (ASB: 5 mg/cm2) and CSE (0.01–1%) for 4 h and then DNA-SB formation was determined as described in the Methods section. Asbestos plus CSE caused DNA-SB formation that was primarily additive in WI-26 cells but synergistic in A549 cells. Data expressed as the mean 6 SEM (WI-26 cells: n 5 7; A549 cells: n 5 6). *p , .05 vs. control; † p , .001 vs. CSE; ¥ p , .05 vs. CSE and/or ASB.
WI-26 cells and the combination of asbestos with CSE (0.1 and 1.0% vol) caused additive WI-26 cell DNA damage. WI-26 cell DNA-SB formation caused by CSE 0.1% alone was comparable to that caused by CSE 0.5% (36.2 6 8.6% vs. 38.0 6 8.2%, respectively [n 5 5]) and no synergistic interaction between asbestos and either dose of CSE was detected (42.9 6 10.5% vs. 41.4 6 12.2%, respectively [n 5 5]). Synergistic WI-26 cell DNA damage was also not detected after exposing the cells to lower doses of asbestos (1 and 2.5 mg/cm2) with CSE (data not shown). We also noted that DNA damage caused by CSE and asbestos in primary isolated RAEC was not synergistic (Fig. 2B). Although SV40 inactivates the p53 gene [27], the levels of DNA-SB in RAEC were similar to the SV40-transformed WI-26 cells (Fig. 4). In contrast to the ATI-like cells, low-dose CSE (0.01%) plus asbestos caused negligible DNA-SB in A549 cells whereas the combination of higher doses of CSE (0.5–1%) plus asbestos induced synergistic DNA damage after 4 h (Fig. 4). Although A549 and WI-26 cell DNA damage after a 4 h exposure to CSE and asbestos
Under basal conditions, GSH levels were more than a log-fold greater in A549 (ATII-like) than WI-26 (type I-like) cells (1.6 6 0.4 vs. 0.1 6 0.05 mg/105 cells, respectively; n 5 3). Asbestos (5 mg/cm2) caused negligible reductions in GSH levels in either WI-26 or A549 cells after 4 h (Fig. 5). CSE diminished GSH levels in WI-26 and A549 cells in a dose-dependent manner. Similar to the effects observed with DNA damage, WI-26 cells were more sensitive to CSE-induced reductions in GSH levels. CSE plus asbestos decreased GSH levels comparable to that noted with CSE alone. Some doses CSE (WI-26 cell: 0.01% and A549 cell: 0.5%) and asbestos caused negligible alterations in GSH levels despite causing DNA damage. These data imply that CSEs, but not low-dose asbestos, reduce GSH levels in AEC. Moreover, the effects of CSE and asbestos on cellular GSH levels are cell type-dependent. Under basal conditions, ATP levels were comparable in A549 (ATII-like) and WI-26 (type I-like) cells (158 6 23 vs. 192 6 63 mg/105 cells, respectively; n 5 5). Asbestos (5 mg/cm2) caused negligible reductions in ATP levels in both cell lines after 18 h (Fig. 6). We previously showed that higher doses of asbestos (25–250 mg/cm2) reduce ATP levels [13]. CSE caused dosedependent decreases in ATP levels in both WI-26 and A549 cells. Similar to DNA damage, WI-26 cells were more sensitive to CSE-induced reductions in ATP than A549 cells. CSE and asbestos decreased ATP levels comparable to that noted with CSE alone. These changes remained significant despite correcting for the ;20% cell death (as assessed by cell number and trypan blue dye exclusion). Of note, some doses CSE (WI-26 cell: 0.01% and A549 cell: 0.5%) and asbestos caused negligible alterations in ATP levels at 18 h despite causing DNA damage at 4 h. We conclude that CSE reduce ATP levels in AEC in a cell type-dependent manner. Antioxidants decrease AEC DNA damage and ATP depletion caused by CSE and asbestos To determine if iron-induced ROS mediate the intracellular effects observed, we assessed whether an iron chelator, phytic acid, or catalase reduced DNA damage or depletion of ATP and GSH levels in A549 cells.
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Fig. 5. CSE and asbestos reduce GSH levels in A549 and WI-26 cells. A549 (left graph) and WI-26 (right graph) cells were exposed to amosite asbestos (ASB: 5 mg/cm2) and (0.01–1%) for 4 h and then GSH levels were determined as described in the Methods section. Data expressed as the mean 6 SEM (WI-26 cells: n 5 3; A549 cells: n 5 3). *p , .05 vs. control.
Phytic acid (500 mM) significantly attenuated A549 cell DNA damage after exposure to CSE and asbestos for 4 h as well as the reductions in ATP levels noted at 18 h (Fig. 7). However, phytic acid did not prevent the reductions in GSH caused by CSE and asbestos after 4 h (Fig. 7). Similar to phytic acid, catalase (100 mg/ml) diminished both DNA-SBs and the reductions in ATP levels but not GSH levels in A549 cells. Therefore, iron-induced free radicals account in part for the DNA damage and reductions in ATP noted in A549 cells exposed to CSE and asbestos. However, other mechanisms likely account for the reductions in GSH caused by CSE and asbestos. In contrast to A549 cells, phytic acid and catalase did not attenuate WI-26 cell DNA damage caused by the combination of asbestos and CSE (data not shown). We also noted that phytic acid did not decrease WI-26 cell cytotoxicity as assessed by specific 51Cr-release after an 18 h exposure period (Control: 0%; asbestos [5 mg/cm2] plus CSE [0.1%]: 23.1 6 6.1%; asbestos [5 mg/cm2] plus CSE [0.1%] plus phytic acid [500 mM]: 29.7 6 5.8%; n 5 6). DISCUSSION
Epidemiological evidence firmly implicate an interactive effect of cigarette smoke and asbestos in causing
bronchogenic carcinoma [1,2]. Despite intensive investigation, the mechanisms involved are not established. Because oxidant-induced alveolar epithelial cell injury is one important event that may account for lung damage caused by cigarette smoke and asbestos [5,6,11,13,14], we explored the effects of these agents on this relevant target cell. In this study, we assessed the cytotoxic effects of aqueous CSE and amosite asbestos on AEC in vitro using two well validated end points: 51Cr-release and DNA-SB formation. We found that the combination of CSE (0.01– 0.1%) and asbestos (5 mg/cm2) caused synergistic injury to two alveolar type I-like cells (WI-26 and RAEC) as assessed by 51Cr-release. Higher doses of each agent caused primarily additive 51Cr-release. CSE augmented asbestos-induced DNA damage synergistically in an ATII-like cell line (A549 cells) but additively or less in the ATI-like cells. CSE plus asbestos also reduced AEC levels of GSH and ATP in a manner that was cell type dependent and not synergistic. Finally, a role for iron-induced ROS was suggested by the finding that phytic acid and catalase each attenuated A549 cell DNA damage as well as the reduction in ATP caused by CSE and asbestos. We observed that the interactive effects of asbestos and CSE on cultured AEC toxicity and DNA damage
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Fig. 6. CSE and asbestos decrease ATP levels in A549 and WI-26 cells. A549 (left graph) and WI-26 (right graph) cells were exposed to amosite asbestos (ASB: 5 mg/cm2) and CSE (0.01–1%) for 4 h and then ATP levels were determined as described in the Methods section. Data expressed as the mean 6 SEM (WI-26 cells: n 5 5; A549 cells: n 5 5). *p , .05 vs. control.
were mainly additive and occasionally comparable to the effect of either agent alone. A synergistic interaction between asbestos and CSE was noted in WI-26 cells and RAEC 51Cr-release after exposure to a single dose of CSE (Figs. 1 and 2) and in A549 cell DNA damage (Fig. 4). The synergistic effects noted herein are less dramatic than an early epidemiological study demonstrating that asbestos workers that smoked had a 5- to ten-fold increased standardized lung cancer death rate as compared to age-matched smokers without asbestos exposure or asbestos workers without cigarette smoke exposure, respectively [1]. However, this study was limited by the small number of asbestos workers that were nonsmokers. A critical review of subsequent studies examining the synergistic interaction between cigarette smoke and asbestos on the incidence of lung cancer suggested that the magnitude of change may not be as large as originally observed [2]. Of the 13 studies reviewed by Seracci [2], four showed an additive or indeterminate interaction between cigarette smoke and asbestos, while nine studies demonstrated a synergistic interaction that varied from a 25 to 75% excess risk due to a simple additive interaction between cigarette smoke and asbestos. Our data with cultured AEC exposed to asbestos and CSE are well within the spectrum observed. Another important factor
that likely accounts for the apparent discrepancy between our data and the early epidemiologic observations is that the clinical expression of synergy between cigarette smoke and asbestos requires many years (typically over 20 years) while our study endpoint is measured over a much shorter time period (4 –18 h). It is, therefore, not surprising that the magnitude of the synergistic interaction observed in this study is less striking than some epidemiological data. Oxidant-induced DNA damage is one mechanism proposed to account for the synergistic interaction between cigarette smoke and asbestos [5,10]. To examine this hypothesis, we utilized fresh CSE that were obtained using techniques that have been widely used by other investigators as a well-validated method to study the effects of cigarette smoke on various biologic functions [6,9,10,23]. The aqueous solutions of whole cigarette smoke used in our study contain both gas- and particulate-phase cigarette smoke. Pryor and coworkers [8,9] demonstrated that the principal radical species in tar consists of conjugated quinones, semiquinones, and hydroquinones. These investigators showed that stable semiquinone radicals are present in aqueous CSE and that these radicals are able to reduce oxygen to superoxide and thereby generate H2O2 and •OH. In contrast to
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Fig. 7. Cigarette smoke extracts and asbestos-induced A549 cell DNA-SB formation and reduction in ATP and GSH levels: Effect of phytic acid and catalase. A549 cells were exposed to cigarette smoke extracts (CSE: 1%) and amosite asbestos (ASB: 5 mg/cm2) with or without phytic acid (500 mM) or catalase (100 mg/ml) and then DNASB, ATP, and GSH were determined as described in the Methods section. Data expressed as the mean 6 SEM (n 5 6). *p , .05 vs. control; † p , .05 vs. CS and asbestos.
the stable radicals in cigarette tar, gas phase cigarette smoke contains short-lived reactive oxygen-centered and carbon-centered radicals. Moreno et al. [28] showed that CSE, similar to that used in the current study, can mobilize redox-active iron from ferritin due in part to hydroquinones and catechols in the CSE. Nakayama and colleagues [6] demonstrated that CSE from one cigarette produce ;104 DNA single strand breaks in A549 cells. One major finding of this study is that CSE and asbestos cause synergistic DNA-SB formation in A549 cells as assessed by fluorescent analysis of DNA unwinding (Fig. 4). Our findings are consistent with data in cell-free systems showing that CSE synergistically augment asbestos-induced DNA-SB [5,10]. The DNA damaging effects of CSE and asbestos appear cell specific because, in contrast to A549 cells, DNA-SB formation was primarily additive in two ATI-like cells, RAEC and WI-26 cells (Figs. 2B and 4). Although A549 cells have a functional p53 gene, the high levels of DNA-SB in WI-26 cells may be due to SV40 T cell antigen inactivation of p53 [27]. This mechanism seems unlikely be-
cause CSE and asbestos caused comparable levels of DNA-SB in primary isolated RAEC and SV40-transformed WI-26 cells. The DNA-SB assay utilized is a sensitive technique that is capable of detecting as few as one break per chromosome [16]. The reduction in fluorescence resulting from compromised ethidium bromideDNA complexes represent nonspecific DNA lesions including strand breaks, oxidized base pairs, and base ligations. Previous studies have demonstrated that this is a sensitive technique to identify DNA damage early after exposure to H2O2 [11,16,17], radiation [17], and asbestos [11]. Our data also suggest that ATI-like cells (WI-26 and RAEC) are more sensitive than ATII-like cells (A549) to DNA damage caused by cigarette smoke and asbestos. DNA damage is an important step in initiating the multistage model of carcinogenesis as well as in facilitating tumor promotion and malignant transformation [29]. The extent of DNA damage detected in AEC exposed to CSE and asbestos depends on the balance of ongoing DNA-SB formation and the efficiency of DNA repair mechanisms. Abnormal cellular DNA is generally efficiently repaired or, if extensive, triggers apoptotic or necrotic cell death. We found that DNA-SB at 4 h were not associated with cell death as detected by trypan blue dye exclusion. Although WI-26 cell death was evident after an 18 h exposure period to CSE and asbestos, it was ;2- to 6-fold less than the levels of DNA-SB detected (Figs. 1 and 4). The type of cell death (e.g., apoptotic or necrotic) was not examined in this study but others have shown that asbestos causes apoptosis that is mediated in part by ROS [30]. It is conceivable that DNA damage in the alveolar epithelium caused by CSE and asbestos could elude proper DNA repair and cell death pathways resulting in the formation of a malignant clone of cells. Considerable evidence, reviewed elsewhere [5], suggest that iron-induced ROS are partially responsible for causing DNA damage after exposure to CSE and asbestos. In this study, we showed that an iron chelator, phytic acid, and catalase, each significantly reduce A549 cell DNA damage and ATP depletion caused by CSE and asbestos. In contrast, neither agent attenuated WI-26 cell DNA damage as well as GSH depletion in both cells. It is possible that the iron bound to the tar components of CSE, such as quinones, are involved in mediating AEC DNA damage. Pryor and coworkers [8,9] demonstrated in a cell-free system that iron chelators, hydroxyl radical scavengers, and catalase each inhibit the formation of DNA nicks and electron spin resonance spectroscopy signals generated by aqueous extracts of cigarette tar. We utilized phytic acid because, just as deferoxamine, it occludes all the reactive coordination sites for iron making it inert, which, subsequently, may protect cells by maintaining iron in the oxidized state [31]. However,
Asbestos and smoke injured epithelial cells
unlike deferoxamine, phytic acid is a safe, natural food additive that does not scavenge ROS [31]. The protective effects of phytic acid noted in this study are comparable to that provided by deferoxamine against asbestos-induced lipid peroxidation, DNA-SB and cellular toxicity in different target cells as well as DNA damage caused by asbestos and CSE in a cell-free system [5,10]. We previously demonstrated that phytic acid, but not ironloaded phytic acid, attenuates asbestos induced WI-26 cell chromium-51 release and the generation of •OH-like species [11]. These findings suggest that the iron chelating properties of phytic acid are important in protecting AEC exposed to asbestos and CSE. These data also implicate iron-catalyzed ROS in causing synergistic DNA damage and ATP depletion in A549 cells after exposure to CSE and asbestos whereas the mechanisms underlying WI-26 cell DNA damage by both agents remain unclear. The lack of a protective effect with an iron chelator in WI-26 cells does not exclude the possibility that ironinduced ROS are involved. Pryor and Stone [8] suggested that metal ions may be firmly attached with tar components or with the DNA thereby preventing access to the chelator. These investigators proposed that tar radicals intimately associated with the DNA generate • OH, which may not be blocked due to the short diffusion radius of the highly reactive •OH. The selective protective effects of phytic acid and catalase in A549 rather than WI-26 cells may also relate to differences in antioxidant defenses. ATII cells, as compared to ATI cells, are more resistant to an oxidant stress that is in part due to differences in their antioxidant defenses [24]. We found that the GSH levels in WI-26 cells are ;10-fold less than in A549 cells (Fig. 6) but that both cells have comparable catalase activity (D. W. Kamp, 1995, unpublished observation). The reduced levels of GSH may render WI-26 cells particularly sensitive to an oxidant stress. The inability of exogenous catalase to protect WI-26 cells may also be due to hydroquinones and aged catechols, two major constituents of CSE that can each inhibit catalase activity and nick DNA [32]. Pryor and colleagues [8,21,33,34] have shown that gas-phase cigarette smoke contains up to 500 ppm of nitric oxide, which can form peroxynitrite and peroxynitrate esters that subsequently reduce cellular thiols and promote apoptotic cell death in lymphocytes. Although we have not identified the specific agent(s) responsible for the effects observed in this study, one constituent in CSE that may mediate both DNA damage and cell death is H2O2. As mentioned previously, CSE can generate H2O2, which, subsequently, could contribute to the interactive effects between the iron in asbestos through Fenton-type chemistry. The protective effects of catalase and phytic acid noted in this study support this
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hypothesis. Another possibility is iron that can accumulate on the tar in CSE. We noted that CSE alone induced dose-dependent AEC DNA damage. Churg and colleagues [4] suggested that iron-catalyzed ROS also enhance asbestos retention in the lungs because catalase and deferoxamine each reduced cigarette smoke-induced epithelial fiber uptake and translocation in tracheal organ cultures. Collectively, these data suggest that AEC DNA damage caused by iron-induced radical formation is one mechanism that accounts for pulmonary toxicity after exposure to asbestos and cigarette smoke. Despite evidence implicating iron-induced ROS, it should be emphasized that other mechanisms may also have an important role in mediating AEC damage after exposure to cigarette smoke and asbestos. First, radicals derived from nitric oxide in the gas-phase of cigarette smoke may also contribute to lung injury [20,33–35]. Gas-phase cigarette smoke causes dose-dependent DNA-SB formation in cultured human bronchial epithelial cells [35]. Although the pattern of DNA base damage by gas-phase cigarette smoke was consistent with attack by •OH, the most common base damage was the formation of xanthine and hypoxanthine presumably due to deamination of guanine and adenosine, respectively, by reactive nitrogen species [35]. A role for reactive nitrogen species in our model is unclear. However, we have observed that inhibitors of inducible nitric oxide synthase do not attenuate asbestos-induced A549 cell DNA damage over 24 h [36]. Second, benzo[a]pyrene, a major carcinogen in cigarette smoke, can form 6-oxobenzo[a]pyrene free radicals that mediate AEC injury and DNA damage. Asbestos catalyzes the oxidation of 6-hydroxy-benzo-[a]pyrene, a metabolite of benzo[a]pyrene, to the 6-oxobenzo[a]pyrene free radical [37]. Additionally, formation of this radical is not inhibited by deferoxamine and occurs equally well in an oxygen-depleted environment [37]. Finally, aqueous CSE also stimulate c-fos expression, a proto-oncogene involved in cell proliferation and apoptosis after exposure to tumor promoters, oxidant stress, GSH depletion, or DNA damage [38]. Notably, the mechanism seems independent of the production of iron-induced ROS because catalase and an iron chelator failed to block c-fos expression despite the fact that these agents significantly prevented DNA-SB formation [38]. Asbestos and cigarette smoke can reduce GSH levels in various pulmonary cells, which may contribute to the pathogenic effect of each agent [13,14,26,39,40]. Depletion of GSH is due in part to inhibition of enzymes involved in GSH synthesis and to enhanced GSH-dependent conjugation reactions and membrane GSH leakage after exposure to aldehydes in cigarette smoke, such as acrolein, formaldehyde and acetaldehyde [14,39]. In contrast to asbestos, we demonstrated that CSE significantly
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reduce GSH levels in A549 and WI-26 cells but were unable to detect a synergistic interaction (Fig. 5). Our data also suggest that the combination of CSE and asbestos deplete GSH levels by mechanisms independent of iron-induced radical formation because phytic acid and catalase were not protective (Fig. 7). The profound reductions in GSH after AEC were exposed to CSE may have promoted cell death. Although not specifically investigated in this study, aldehydes in cigarette smoke deplete GSH in bronchial epithelial cells by mechanisms that are not coupled to oxidative stress [39]. As mentioned, peroxynitrite can also deplete cellular GSH and contribute to cell death [8,20,33]. ATP depletion is one of the many biochemical events that can occur in cells exposed to ROS and, if severe, can also augment cell death [17,29]. We were unable to detect a synergistic reduction in A549 and WI-26 cell ATP levels after exposure to CSE and asbestos. However, we found that CSE plus asbestos induces DNA-SB formation without necessarily reducing ATP and GSH levels in AEC (Figs. 4, 5, and 6). Therefore, an abnormal clone of cells may originate if either the DNA repair mechanisms or cell death pathways are incomplete. In summary, we have demonstrated that CSE and asbestos can cause DNA-SB formation and cell death in cultured AEC, relevant target cells of both agents. While the interactive effects between CSE and asbestos were primarily additive, synergistic DNA damage and cell death were noted under some conditions. Our results indicate that iron-induced free radicals contribute to A549 cell DNA damage and ATP depletion and that DNA damage can occur without necessarily altering the levels of ATP or GSH. In contrast to A549 cells, DNA damage caused by CSE and asbestos in ATI-like cells (WI-26 and RAEC) was not synergistic nor was phytic acid or catalase protective. We also showed that CSE significantly reduced GSH levels in A549 and WI-26 cells and that phytic acid and catalase did not reduce these effects. We conclude that the mechanism causing alveolar epithelial cell toxicity after exposure to cigarette smoke and asbestos is due in part to DNA damage by iron-induced free radicals. The DNA damaging effects of both agents depend on the cell type exposed and are not necessarily associated with alterations in cellular energy levels or GSH. Although the relevance of our in vitro findings require additional study, Churg and associates [41] demonstrated in rats that amosite asbestos and cigarette smoke induce a brief significant synergistic increase in the DNA labeling index in small airway epithelial cells but not in vascular or mesothelial cells. We speculate that one mechanism that accounts for the enhanced incidence of bronchogenic carcinoma after exposure to cigarette smoke and asbestos is due to iron-
induced ROS that damage DNA in the pulmonary epithelium. Acknowledgements—This work was supported in part by a grant from the Department of Veterans Affairs (Merit Review) and the Asbestos Victims Special Fund Trust, a publicly supported charitable organization. The contents of this article do not necessarily reflect the views of the Trust or the parties that support it. The authors are grateful for the technical assistance provided by Chen Zhang and Elsa Escalara.
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ABBREVIATIONS
AEC—alveolar epithelial cells GSH— glutathione CSE— cigarette smoke extracts • OH— hydroxyl radicals ROS—reactive oxygen species DNA-SB—DNA strand breaks AOE—antioxidant enzymes PBS—phosphate buffered saline H2O2— hydrogen peroxide ATII—alveolar type II DMEM—Dulbecco’s modified Eagle medium RAEC—rat alveolar type II cell cultured for 7–10 d FBS—fetal bovine serum HBSS—Hanks balance salt solution plus calcium, magnesium and 15 mM N-2-hydroxy-ethylpiperazine-N92-ethanesulfonic acid DMSO— dimethylsulfoxide
PII S0891-5849(98)00158-0
Original Contribution CIGARETTE SMOKE AUGMENTS ASBESTOS-INDUCED ALVEOLAR EPITHELIAL CELL INJURY: ROLE OF FREE RADICALS DAVID W. KAMP,* MARC J. GREENBERGER,* JANE S. SBALCHIERRO, SCOTT E. PREUSEN,* SIGMUND A. WEITZMAN†
and
Department of Medicine, pDivision of Pulmonary Medicine, and †Hematology–Oncology, Northwestern University Medical School and Veterans Affairs Chicago Health Care System, Lakeside Division, Chicago, IL, USA (Received 16 March 1998; Revised 12 June 1998; Accepted 12 June 1998)
Abstract—Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not established. Alveolar epithelial cell (AEC) injury due to oxidant-induced DNA damage and depletion of glutathione (GSH) and adenosine triphosphate (ATP) may be one important mechanism. We previously showed that amosite asbestosinduces hydroxyl radical production and DNA damage to cultured AEC and that phytic acid, an iron chelator, is protective. We hypothesized that whole cigarette smoke extracts (CSE) augment amosite asbestos-induced AEC injury by generating iron-induced free radicals that damage DNA and reduce cellular GSH and ATP levels. Asbestos or CSE each caused dose-dependent toxicity to AEC (WI-26 and rat alveolar type I-like cells) as assessed by 51chromium release. The combination of asbestos (5 mg/cm2) and CSE (0.01– 0.1%) caused synergistic injury whereas higher doses of each agent primarily had an additive toxic effect. Asbestos (5 mg/cm2) augmented CSE-induced (0.01–1.0%) AEC DNA damage over a 4 h exposure period as assessed by an alkaline unwinding, ethidium bromide fluorometric technique. These effects were synergistic in A549 cells and additive in WI-26 cells. Asbestos (5 mg/cm2) and CSE (0.5–1.0%) reduced A549 and WI-26 cell GSH levels as assessed spectrophotometrically and ATP levels as assessed by luciferin/luciferase chemiluminescence but a synergistic interaction was not detected. Phytic acid (500 mM) and catalase (100 mg/ml) each attenuated A549 cell DNA damage and depletion of ATP caused by asbestos and CSE. However, neither agent attenuated WI-26 cell DNA damage nor the reductions in GSH levels in WI-26 and A549 cells exposed to asbestos and CSE. We conclude that CSE enhance asbestos-induced DNA damage in cultured alveolar epithelial cells. These data provide additional support that asbestos and cigarette smoke are genotoxic to relevant target cells in the lung and that iron-induced free radicals may in part cause these effects. © 1998 Elsevier Science Inc. Keywords—Asbestos, Cigarette smoke, Alveolar epithelial cells, DNA damage, Reactive oxygen species, Lung injury, Glutathione, ATP, Free radical
INTRODUCTION
Alveolar epithelial cell (AEC) injury is one important event that may perpetuate lung damage caused by asbestos and cigarette smoke. Asbestos is internalized by alveolar type I cells resulting in cytotoxicity and triggering of a proliferative response in alveolar type II (ATII) cells [3]. Studies in animals and humans demonstrate that cigarette smoke promotes the pathogenic effects of asbestos by impairing lung clearance of the fibers in part by augmenting asbestos uptake across the pulmonary epithelium [4]. Several lines of evidence strongly implicate reactive oxygen species (ROS) generated by cigarette smoke and asbestos as one important mechanism causing synergistic lung damage. First, cigarette smoke and asbestos each generates significant levels of ROS [5–7]. Cigarette
Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not yet established [1,2]. Although epidemiologic evidence attributes bronchogenic carcinoma to a multiplicative interaction between tobacco smoke and asbestos, a simple additive response has also been reported [1,2]. Despite extensive investigations exploring the interactive effects between cigarette smoke and asbestos, the precise mechanisms involved at the cellular and molecular level are unclear. Address correspondence to: David W. Kamp, M.D., Northwestern University Medical School, Pulmonary Division; Passavant, Room 777, 303 East Superior Street, Chicago, IL 60611, USA; Tel: (312) 908-8163; Fax: (312) 908-4650; E-Mail: [email protected] 728
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smoke is a complex mixture containing over 5000 different substances [8]. Cigarette tar, which contains at least 1017 free radicals per gram [8], is a stable substance that can accumulate iron that promotes the formation of highly reactive hydroxyl radicals (•OH) [8,9]. The gas phase of cigarette smoke also contains short-lived ROS that are potent oxidants [5]. Iron-induced •OH are implicated in causing genotoxicity in cells exposed to aqueous whole cigarette smoke extracts (CSE). Ferrous iron in asbestos in the presence of hydrogen peroxide (H2O2) also produces •OH via a Fenton reaction in the following manner [7]:
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duced AEC injury in vitro by generating iron-catalyzed free radicals that damage DNA and reduce cellular GSH and ATP levels. We demonstrate that CSE increase amosite asbestos-induced cytotoxicity and DNA-SB formation in cultured AEC. In addition, synergistic DNA damage was noted in A549 cells and these effects were attenuated by phytic acid and catalase. Both agents caused AEC DNA damage without necessarily altering GSH and ATP. METHODS
Mineral dusts H2O2 1 Asbestos-Fe21 3 •OH1OH1 Asbestos-Fe31 Jackson and coworkers [10] demonstrated that CSE synergistically augment asbestos-induced •OH formation detected by electron paramagnetic resonance spectroscopy and bacteriophage PM2 DNA strand break (DNASB) formation that were both prevented by •OH scavengers or iron chelators. Second, cigarette smoke-induced fiber uptake by pulmonary epithelial cells is diminished by antioxidant enzymes (AOE) or iron chelators [4]. Third, AOE, such as catalase and superoxide dismutase, or iron chelators each attenuate asbestos-induced AEC injury by reducing lipid peroxidation, DNA-SB formation, and 8-OH-deoxyguanosine base pair changes in DNA [5,7,10 –12]. Exogenous AOE and reduced glutathione (GSH) also diminish cigarette smoke-induced A549 cell DNA damage and altered permeability [13, 14]. Fourth, as recently reviewed [5], ROS generated by inflammatory cells in vitro and in vivo may contribute to pulmonary toxicity. Finally, asbestos and other oxidant stresses alter the expression of AOE in the pulmonary epithelium suggesting important adaptive responses [15]. Although these data firmly support the hypothesis that ROS are one mechanism that cigarette smoke and asbestos synergistically injure the lungs, the effects of these agents on an important target cell, AEC, are unknown. The cellular and molecular events culminating in AEC injury after an oxidant stress from asbestos and cigarette smoke are uncertain. DNA strand breaks are one of the earliest cellular changes occurring after an oxidant stress from H2O2 or radiation [16,17]. ROSinduced DNA damage can subsequently deplete GSH and ATP levels resulting in cell death [5,14,17]. Cigarette smoke or asbestos each alone cause DNA base pair modifications and strand break formation [6,13,18 –21]. We previously showed that asbestos-induced AEC toxicity is due in part to DNA-SB formation caused by the production of iron-induced •OH-like species [11]. We hypothesized that CSE augment amosite asbestos-in-
Amosite asbestos used in these experiments was Union International Contre Le Cancer (UICC) Reference Standard samples kindly supplied by Dr. V. Timbrell [22]. A stock concentration of amosite (5 mg/ml) was prepared in Hanks balanced salt solution plus calcium, magnesium and 15 mM N-2-hydroxyethylpiperazine-N92-ethanesulfonic acid (HBSS). The suspension was autoclaved and stored at 4°C. Before use, the mineral dusts were warmed to 37°C and vigorously vortexed to ensure a uniform suspension. Cigarette smoke extract preparation Cigarette smoke extracts were prepared fresh for each experiment as previously described [10,23]. Briefly, CSE were obtained by bubbling cigarette smoke from 1 commercial unfiltered cigarette (Pall Mall) through phosphate buffered saline (PBS) (10 ml) at 3 puffs/min. It is estimated that the cigarette smoke extract concentration, which simulates the concentration present in adult smoker’s lung, is approximately 0.1% (vol/vol) [23]. As a result, AEC were exposed to various dilutions of CSE (0.01–1.0% vol/vol) for variable periods with and without asbestos present. Because Holden and colleagues [23] showed that CSE containing 1 cigarette/ml (100% vol/vol) have approximately 1 mg/ml of nicotine, we estimated that the final concentration of nicotine in the cultures varied from 0.1–10 mg/ml. Alveolar epithelial cells SV-40 transformed WI-26 cells (VA4; CCL-95.1) and A549 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). WI-26 cells are derived from human embryonal lung. Electron microscopic analysis of WI-26 cells in our laboratory have revealed type-1 pneumocyte-like morphology in that they have tight junctions and no lamellar bodies. WI-26 cells were maintained in Eagles minimal essential medium (GIBCO, Grand Island, NY, USA) with L-
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glutamine (0.3 mg/ml), nonessential amino acids, penicillin (100 U/ml), streptomycin (200 mg/ml), and amphotericin B (0.25 mg/ml), and supplemented with 20% fetal bovine serum (FBS; GIBCO) and grown in 5% CO2 at 37°C. A549 cells, which are human bronchoalveolar carcinoma-derived cells with synthetic and morphologic characteristics of alveolar epithelial type II cells, were maintained in Dulbecco’s modification of Dulbecco’s modified Eagle medium (DMEM) supplemented as above only with 10% FBS. Rat ATII cells were isolated from specific pathogenfree adult Harlan–Sprague–Dawley rats (200 –250 g) as previously described [15,17]. Briefly, the cells were obtained from rat lungs by elastase digestion, followed by mincing and sequential filtration. ATII cell purity was enhanced by “panning” the cell suspension on plates coated with rat immunoglobulin G (IgG 500 mg/ml; Sigma). The cell suspension was then washed, resuspended in DMEM plus 10% FBS with antibiotics, and plated at a density of 5 3 105 cells/well in a 24-well plate (Costar, Cambridge, MA, USA). After isolation, cell viability was .90% by trypan dye exclusion and type II cell purity 24 h after isolation was 80 to 85% ATII as assessed by a modified Papanicolaou stain (n 5 4). The cells were incubated in 95% air/5% CO2 at 37°C for 7 to 10 days before use. Alveolar type II cells cultured for 7 to 10 days (RAEC) gradually lose their characteristic features (i.e., lamellar bodies and surfactant production) and morphologically resemble alveolar type I cells [24]. RAEC were chosen for these experiments because type I cells comprise nearly 95% of the alveolar surface area and asbestos is noted in these cells early after exposure [3]. Previous studies in our laboratory have shown that cell purity assessed by tannic acid staining of lamellar bodies varies with time in culture (% lamellar body staining: 24 h: 86 6 1%; 48 h: 80 6 3%; 5 d: 37 6 2%; and 7–10 d: 6 6 2%; n 5 3–6 at each time point) and that over 95% of the cells contain cytokeratin by indirect immunoflouresence with anticytokeratin antibody after 5 to 7 days in culture [25]. Cytotoxicity assay Cytotoxicity was measured by a 51Cr release assay previously described by our laboratory with some minor modifications [11,25]. Briefly, WI-26 cells were grown to confluency in gelatin-coated 24 well (2 cm2) tissue culture plates, washed twice with sterile HBSS, radiolabeled with 1.0 mCi/well Na251CrO4 (ICN Pharmaceuticals, Irvine, CA, USA) for 18 h in media supplemented with 2% FBS and then washed three times with HBSS to remove unbound 51Cr. The radiolabeled WI-26 cells were then incubated with one of the test conditions in HBSS. In some experiments, catalase or phytic acid
(Sigma) was added prior to the CSE and asbestos. After incubation for 18 h at 37°C in 95% air/5% CO2, the medium bathing the WI-26 cells (1 ml) was aspirated and the WI-26 cells were washed three times with 0.5 ml of HBSS. An aliquot of the combined aspirate and wash (Media) was placed into a test tube and counted in a gamma counter (Beckman Instruments, Palo Alto, CA, USA). The total possible counts (Total) were determined from control cell lysates (1 ml 1N NH4 OH 3 10 min) plus control Media. Injury to the WI-26 cells under each experimental condition was then calculated by the percent of 51Cr release defined as: $~test Media cpm 2 control Media cpm!/total cpm%) 3 100. This cytotoxicity index represents cell lysis and detachment corrected for background spontaneous 51Cr release from control WI-26 cells assayed the same day. Spontaneous 51Cr release from WI-26 cells incubated 18 h under control conditions (HBSS) was 30.5 6 2.6% (n 5 15). Cell number (data not shown), viability ($90% trypan blue dye exclusion), and morphology were similar in WI-26 cells maintained under control conditions compared with cells maintained in media supplemented with FBS. DNA Strand Break (DNA-SB) The formation of DNA-SB were assessed by alkaline unwinding and ethidium bromide fluorescence, as previously described [11]. Because ethidium bromide preferentially binds to double stranded DNA in alkali, the relative amounts of nonbroken double-stranded DNA and broken single-stranded DNA can be assessed [17]. The alveolar epithelial cells (WI-26, A549, and RAEC) were exposed to various concentrations of CSE and asbestos for variable periods. In some experiments, phytic acid or catalase was added prior to the CSE and asbestos. Following incubation with asbestos, the cells were washed three times with HBSS buffer and then analyzed as follows: 100 ml of solution B (0.25 M mesoinositol, 10 mM sodium phosphate, 1 mM MgCl2; pH 7.2) followed without mixing with 100 ml solution C (9 M urea, 10 mM NaOH, 2.5 M cyclohexanediamineacetate and 0.1% sodium dodecyl sulfate) were added to each well and then incubated at 0°C for 10 min to enable cell lysis and chromatin disrupture. Without mixing, 50 ml of solution D (0.45 vol solution C in 0.2 N NaOH) and 50 ml solution E (0.40 vol solution C and 2 N NaOH) were added and incubated at 0°C for 30 min. After incubation, 200 ml of solution F (1M glucose and 14 mM mercaptoethanol) was added to each well followed by
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sonication (Sonifier Cell Disrupter; Heat Systems Ultrasonics, Plainview, NY, USA) for 3 s and addition of 750 ml of solution G (6.7 mg/ml ethidium bromide in 13.3 mM NaOH). Fluorescence was then determined using a Sequoia Turner fluorometer (Model 450; Mountain View, CA, USA) with excitation at 520 nm and emission at 585 nm. The percentage of double stranded DNA ~% ds-DNA) 5 (F 2 Fmin)/(Fmax 2 Fmin) 3 100
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Glutathione assay Total glutathione was determined as previously described [26]. The assay is based on the rate of decrease of 5-59-dinitrobis(-2-nitrobenzoic acid) measured spectrophotometrically at 412 nm. AEC were exposed for 4 h at 37°C to various concentrations of CSE with or without asbestos before GSH levels were determined and expressed per cell number.
ATP Measurement where F is the fluorescence of the experimental condition, Fmin the background ethidium bromide fluorescence determined after converting all the DNA into single strand form, and Fmax is the fluorescence determined after adding the mercaptoethanol solution before the alkaline solution to maintain the pH 11.0, which is well below that needed to augment unwinding of single stranded DNA. Results are expressed as DNA-SB defined as 100 2 % ds-DNA. The amount of single stranded DNA present in alkali after sonication is proportional to the number of DNA-SB (either single or double) because DNA-SB varies directly as a function of the extent of DNA unwinding [10]. We previously showed that the amount of asbestos used in these experiments (10 mg/ml or 5 mg/cm2) does not quench the fluorescent signal nor cause DNA damage while harvesting the cells [11].
Cellular ATP levels were assessed by the luciferin/ luciferase chemiluminescence technique, as previously described [26]. Confluent WI-26 and A549 cells were washed three times, and then exposed to various concentrations of CSE with or without asbestos (10 mg/ml). After incubation for 18 h at 37°C, the cells were washed three times, heated to 95°C for 4 min in 2 ml of KH2PO4 and 4 mM MgPO4 and placed on ice. Within 4 h of sample collection, ATP levels were be assayed by combining 1 ml of sample with 2 ml of 50 mM NaAsO2, 20 mM MgSO4 and 50 ml of luciferin/luciferase. Light emission was quantified precisely 15 s after the addition of luciferin/luciferase. ATP levels were determined from standards prepared in parallel dishes not containing AEC but otherwise handled in a similar manner as described. This assay has been shown to be linear for 1029 to 1025 M ATP. The data are expressed as nM ATP per 106 cells.
Fig. 1. Cigarette smoke extracts (CSE) and amosite asbestos (ASB) injure WI-26 cells. WI-26 cells were exposed to CSE (0.01 to 1.0%) and/or ASB (5 mg/cm2) for 18 h. Cell cytotoxicity was assessed based upon the percentage of 51Cr-released corrected for the spontaneous release from control cells as defined in the Methods section. Asbestos-induced WI-26 cell cytotoxicity was synergistically increased by low-dose CSE (0.01%) and additively increased at higher doses of CSE (0.1–1.0%). Data expressed as the mean 6 SEM (n 5 8). *p , .01 vs. ASB and/or CSE; † p , .05 vs. control; § p , .001 vs. control.
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Statistical Analysis The results of each experimental condition were determined from the mean of duplicate or triplicate trials. Unless otherwise noted, at least 6 separate experiments (n 5 6) were performed for each group of experiments and the data expressed as the mean 6 SEM. A twotailed, unpaired Student’s t-test was used to assess significance between two groups of data. Analysis of variance was used when comparing more than two groups; differences between two groups within the set were analyzed by a Fischer’s protected least significant difference test. A synergistic effect was defined when the observed effect caused by CSE and asbestos was greater than the sum of each agent. p-values , .05 were considered significant. RESULTS
cific 51Cr-release after an 18 h exposure period. CSE resulted in dose-dependent toxicity to WI-26 cells. The combination of CSE (0.1 and 1.0% vol) and asbestos (5 mg/cm2) had an additive cytotoxic effect. However, synergistic WI-26 cells injury occurred with CSE (0.01% vol) and asbestos ( p , .01 vs. asbestos plus CSE). Higher concentrations of asbestos (25–250 mg/cm2) with CSE caused primarily additive toxic effects (data not shown). As shown in Fig. 2A, amosite asbestos (5 mg/ cm2) alone caused modest injury to primary isolated RAEC after an 18 h exposure period (6% Sp 51Crrelease; p , .05). Similar to WI-26 cells, CSE (0.1% vol) plus asbestos (5 mg/cm2) also induced synergistic injury to RAEC (Fig. 2A). Therefore, CSE augments amosite asbestos-induced injury to WI-26 and RAEC cells. CSE augment asbestos-induced AEC DNA Damage
CSE increase asbestos-induced AEC cytotoxicity As shown in Fig. 1, amosite asbestos (5 mg/cm ) alone caused negligible injury to a human pulmonary epithelial-like cell line (WI-26 cells) as assessed by spe2
Fig. 2. (A) Cigarette smoke extracts (CSE) and amosite asbestos (ASB) synergistically injure RAEC cells. RAEC cells were exposed to CSE (0.1%) and/or ASB (5 mg/cm2) for 18 h. Cell cytotoxicity was assessed based upon the percentage of 51Cr-release corrected for the spontaneous release as defined in the Methods section. Data expressed as the mean 6 SEM (n 5 6). (B) Cigarette smoke extracts (CSE) and amosite asbestos (ASB) cause DNA damage in RAEC cells. RAEC cells were exposed to CSE (0.1%) and/or ASB (5 mg/cm2) for 4 h. DNA-SB formation was assessed as defined in the Methods section. Data expressed as the mean 6 SEM (n 5 5). *p , .05 vs. control; † p , .02 vs. ASB alone, CSE alone, or ASB plus CSE.
We determined whether CSE and asbestos induce AEC DNA damage. As shown in Fig. 3, CSE alone caused dose-dependent WI-26 cell DNA-SB formation after a 4 h exposure period (r 5 0.94; p , .05). In contrast to these type I-like cells, CSE induced minimal levels of DNA-SB formation in A549 cells, a cell line possessing type II cell features. Significant A549 cell DNA damage was noted only after exposure to the highest dose of CSE tested (Fig. 3). Therefore, CSE cause DNA damage to AEC and these effects are cell typedependent. As illustrated in Fig. 4, CSE (0.01–1.0%) and asbestos (5 mg/cm2) each alone induced DNA-SB formation in
Fig. 3. DNA strand breaks induced by CSE in WI-26 and A549 cells. WI-26 and A549 cells were exposed to CSE (0 –1.0%) for 4 h and then the percentage of DNA-SBs were determined by the alkaline unwinding, ethidium bromide fluorometric technique described in the Methods section. CSE caused dose-dependent DNA-SB formation in WI-26 cells (solid bars) and lesser amounts of DNA-SB in A549 cells (stippled bars). Data expressed as the mean 6 SEM (WI-26 cells: n 5 7; A549 cells: n 5 6). *p , .05 vs. control.
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occurred with negligible changes in cell number or viability (data not shown), cytotoxicity was noted by 18 h (Figs. 1 and 2A). We conclude that CSE plus asbestos induce DNA damage in WI-26 cells and RAEC that is less than or comparable to an additive effect of each agent alone but is synergistic in A549 cells. CSE reduce GSH and ATP levels in AEC
Fig. 4. CSE and asbestos cause DNA-SB in WI-26 and A549 cells. WI-26 (top graph) and A549 (bottom graph) cells were exposed to amosite asbestos (ASB: 5 mg/cm2) and CSE (0.01–1%) for 4 h and then DNA-SB formation was determined as described in the Methods section. Asbestos plus CSE caused DNA-SB formation that was primarily additive in WI-26 cells but synergistic in A549 cells. Data expressed as the mean 6 SEM (WI-26 cells: n 5 7; A549 cells: n 5 6). *p , .05 vs. control; † p , .001 vs. CSE; ¥ p , .05 vs. CSE and/or ASB.
WI-26 cells and the combination of asbestos with CSE (0.1 and 1.0% vol) caused additive WI-26 cell DNA damage. WI-26 cell DNA-SB formation caused by CSE 0.1% alone was comparable to that caused by CSE 0.5% (36.2 6 8.6% vs. 38.0 6 8.2%, respectively [n 5 5]) and no synergistic interaction between asbestos and either dose of CSE was detected (42.9 6 10.5% vs. 41.4 6 12.2%, respectively [n 5 5]). Synergistic WI-26 cell DNA damage was also not detected after exposing the cells to lower doses of asbestos (1 and 2.5 mg/cm2) with CSE (data not shown). We also noted that DNA damage caused by CSE and asbestos in primary isolated RAEC was not synergistic (Fig. 2B). Although SV40 inactivates the p53 gene [27], the levels of DNA-SB in RAEC were similar to the SV40-transformed WI-26 cells (Fig. 4). In contrast to the ATI-like cells, low-dose CSE (0.01%) plus asbestos caused negligible DNA-SB in A549 cells whereas the combination of higher doses of CSE (0.5–1%) plus asbestos induced synergistic DNA damage after 4 h (Fig. 4). Although A549 and WI-26 cell DNA damage after a 4 h exposure to CSE and asbestos
Under basal conditions, GSH levels were more than a log-fold greater in A549 (ATII-like) than WI-26 (type I-like) cells (1.6 6 0.4 vs. 0.1 6 0.05 mg/105 cells, respectively; n 5 3). Asbestos (5 mg/cm2) caused negligible reductions in GSH levels in either WI-26 or A549 cells after 4 h (Fig. 5). CSE diminished GSH levels in WI-26 and A549 cells in a dose-dependent manner. Similar to the effects observed with DNA damage, WI-26 cells were more sensitive to CSE-induced reductions in GSH levels. CSE plus asbestos decreased GSH levels comparable to that noted with CSE alone. Some doses CSE (WI-26 cell: 0.01% and A549 cell: 0.5%) and asbestos caused negligible alterations in GSH levels despite causing DNA damage. These data imply that CSEs, but not low-dose asbestos, reduce GSH levels in AEC. Moreover, the effects of CSE and asbestos on cellular GSH levels are cell type-dependent. Under basal conditions, ATP levels were comparable in A549 (ATII-like) and WI-26 (type I-like) cells (158 6 23 vs. 192 6 63 mg/105 cells, respectively; n 5 5). Asbestos (5 mg/cm2) caused negligible reductions in ATP levels in both cell lines after 18 h (Fig. 6). We previously showed that higher doses of asbestos (25–250 mg/cm2) reduce ATP levels [13]. CSE caused dosedependent decreases in ATP levels in both WI-26 and A549 cells. Similar to DNA damage, WI-26 cells were more sensitive to CSE-induced reductions in ATP than A549 cells. CSE and asbestos decreased ATP levels comparable to that noted with CSE alone. These changes remained significant despite correcting for the ;20% cell death (as assessed by cell number and trypan blue dye exclusion). Of note, some doses CSE (WI-26 cell: 0.01% and A549 cell: 0.5%) and asbestos caused negligible alterations in ATP levels at 18 h despite causing DNA damage at 4 h. We conclude that CSE reduce ATP levels in AEC in a cell type-dependent manner. Antioxidants decrease AEC DNA damage and ATP depletion caused by CSE and asbestos To determine if iron-induced ROS mediate the intracellular effects observed, we assessed whether an iron chelator, phytic acid, or catalase reduced DNA damage or depletion of ATP and GSH levels in A549 cells.
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Fig. 5. CSE and asbestos reduce GSH levels in A549 and WI-26 cells. A549 (left graph) and WI-26 (right graph) cells were exposed to amosite asbestos (ASB: 5 mg/cm2) and (0.01–1%) for 4 h and then GSH levels were determined as described in the Methods section. Data expressed as the mean 6 SEM (WI-26 cells: n 5 3; A549 cells: n 5 3). *p , .05 vs. control.
Phytic acid (500 mM) significantly attenuated A549 cell DNA damage after exposure to CSE and asbestos for 4 h as well as the reductions in ATP levels noted at 18 h (Fig. 7). However, phytic acid did not prevent the reductions in GSH caused by CSE and asbestos after 4 h (Fig. 7). Similar to phytic acid, catalase (100 mg/ml) diminished both DNA-SBs and the reductions in ATP levels but not GSH levels in A549 cells. Therefore, iron-induced free radicals account in part for the DNA damage and reductions in ATP noted in A549 cells exposed to CSE and asbestos. However, other mechanisms likely account for the reductions in GSH caused by CSE and asbestos. In contrast to A549 cells, phytic acid and catalase did not attenuate WI-26 cell DNA damage caused by the combination of asbestos and CSE (data not shown). We also noted that phytic acid did not decrease WI-26 cell cytotoxicity as assessed by specific 51Cr-release after an 18 h exposure period (Control: 0%; asbestos [5 mg/cm2] plus CSE [0.1%]: 23.1 6 6.1%; asbestos [5 mg/cm2] plus CSE [0.1%] plus phytic acid [500 mM]: 29.7 6 5.8%; n 5 6). DISCUSSION
Epidemiological evidence firmly implicate an interactive effect of cigarette smoke and asbestos in causing
bronchogenic carcinoma [1,2]. Despite intensive investigation, the mechanisms involved are not established. Because oxidant-induced alveolar epithelial cell injury is one important event that may account for lung damage caused by cigarette smoke and asbestos [5,6,11,13,14], we explored the effects of these agents on this relevant target cell. In this study, we assessed the cytotoxic effects of aqueous CSE and amosite asbestos on AEC in vitro using two well validated end points: 51Cr-release and DNA-SB formation. We found that the combination of CSE (0.01– 0.1%) and asbestos (5 mg/cm2) caused synergistic injury to two alveolar type I-like cells (WI-26 and RAEC) as assessed by 51Cr-release. Higher doses of each agent caused primarily additive 51Cr-release. CSE augmented asbestos-induced DNA damage synergistically in an ATII-like cell line (A549 cells) but additively or less in the ATI-like cells. CSE plus asbestos also reduced AEC levels of GSH and ATP in a manner that was cell type dependent and not synergistic. Finally, a role for iron-induced ROS was suggested by the finding that phytic acid and catalase each attenuated A549 cell DNA damage as well as the reduction in ATP caused by CSE and asbestos. We observed that the interactive effects of asbestos and CSE on cultured AEC toxicity and DNA damage
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Fig. 6. CSE and asbestos decrease ATP levels in A549 and WI-26 cells. A549 (left graph) and WI-26 (right graph) cells were exposed to amosite asbestos (ASB: 5 mg/cm2) and CSE (0.01–1%) for 4 h and then ATP levels were determined as described in the Methods section. Data expressed as the mean 6 SEM (WI-26 cells: n 5 5; A549 cells: n 5 5). *p , .05 vs. control.
were mainly additive and occasionally comparable to the effect of either agent alone. A synergistic interaction between asbestos and CSE was noted in WI-26 cells and RAEC 51Cr-release after exposure to a single dose of CSE (Figs. 1 and 2) and in A549 cell DNA damage (Fig. 4). The synergistic effects noted herein are less dramatic than an early epidemiological study demonstrating that asbestos workers that smoked had a 5- to ten-fold increased standardized lung cancer death rate as compared to age-matched smokers without asbestos exposure or asbestos workers without cigarette smoke exposure, respectively [1]. However, this study was limited by the small number of asbestos workers that were nonsmokers. A critical review of subsequent studies examining the synergistic interaction between cigarette smoke and asbestos on the incidence of lung cancer suggested that the magnitude of change may not be as large as originally observed [2]. Of the 13 studies reviewed by Seracci [2], four showed an additive or indeterminate interaction between cigarette smoke and asbestos, while nine studies demonstrated a synergistic interaction that varied from a 25 to 75% excess risk due to a simple additive interaction between cigarette smoke and asbestos. Our data with cultured AEC exposed to asbestos and CSE are well within the spectrum observed. Another important factor
that likely accounts for the apparent discrepancy between our data and the early epidemiologic observations is that the clinical expression of synergy between cigarette smoke and asbestos requires many years (typically over 20 years) while our study endpoint is measured over a much shorter time period (4 –18 h). It is, therefore, not surprising that the magnitude of the synergistic interaction observed in this study is less striking than some epidemiological data. Oxidant-induced DNA damage is one mechanism proposed to account for the synergistic interaction between cigarette smoke and asbestos [5,10]. To examine this hypothesis, we utilized fresh CSE that were obtained using techniques that have been widely used by other investigators as a well-validated method to study the effects of cigarette smoke on various biologic functions [6,9,10,23]. The aqueous solutions of whole cigarette smoke used in our study contain both gas- and particulate-phase cigarette smoke. Pryor and coworkers [8,9] demonstrated that the principal radical species in tar consists of conjugated quinones, semiquinones, and hydroquinones. These investigators showed that stable semiquinone radicals are present in aqueous CSE and that these radicals are able to reduce oxygen to superoxide and thereby generate H2O2 and •OH. In contrast to
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Fig. 7. Cigarette smoke extracts and asbestos-induced A549 cell DNA-SB formation and reduction in ATP and GSH levels: Effect of phytic acid and catalase. A549 cells were exposed to cigarette smoke extracts (CSE: 1%) and amosite asbestos (ASB: 5 mg/cm2) with or without phytic acid (500 mM) or catalase (100 mg/ml) and then DNASB, ATP, and GSH were determined as described in the Methods section. Data expressed as the mean 6 SEM (n 5 6). *p , .05 vs. control; † p , .05 vs. CS and asbestos.
the stable radicals in cigarette tar, gas phase cigarette smoke contains short-lived reactive oxygen-centered and carbon-centered radicals. Moreno et al. [28] showed that CSE, similar to that used in the current study, can mobilize redox-active iron from ferritin due in part to hydroquinones and catechols in the CSE. Nakayama and colleagues [6] demonstrated that CSE from one cigarette produce ;104 DNA single strand breaks in A549 cells. One major finding of this study is that CSE and asbestos cause synergistic DNA-SB formation in A549 cells as assessed by fluorescent analysis of DNA unwinding (Fig. 4). Our findings are consistent with data in cell-free systems showing that CSE synergistically augment asbestos-induced DNA-SB [5,10]. The DNA damaging effects of CSE and asbestos appear cell specific because, in contrast to A549 cells, DNA-SB formation was primarily additive in two ATI-like cells, RAEC and WI-26 cells (Figs. 2B and 4). Although A549 cells have a functional p53 gene, the high levels of DNA-SB in WI-26 cells may be due to SV40 T cell antigen inactivation of p53 [27]. This mechanism seems unlikely be-
cause CSE and asbestos caused comparable levels of DNA-SB in primary isolated RAEC and SV40-transformed WI-26 cells. The DNA-SB assay utilized is a sensitive technique that is capable of detecting as few as one break per chromosome [16]. The reduction in fluorescence resulting from compromised ethidium bromideDNA complexes represent nonspecific DNA lesions including strand breaks, oxidized base pairs, and base ligations. Previous studies have demonstrated that this is a sensitive technique to identify DNA damage early after exposure to H2O2 [11,16,17], radiation [17], and asbestos [11]. Our data also suggest that ATI-like cells (WI-26 and RAEC) are more sensitive than ATII-like cells (A549) to DNA damage caused by cigarette smoke and asbestos. DNA damage is an important step in initiating the multistage model of carcinogenesis as well as in facilitating tumor promotion and malignant transformation [29]. The extent of DNA damage detected in AEC exposed to CSE and asbestos depends on the balance of ongoing DNA-SB formation and the efficiency of DNA repair mechanisms. Abnormal cellular DNA is generally efficiently repaired or, if extensive, triggers apoptotic or necrotic cell death. We found that DNA-SB at 4 h were not associated with cell death as detected by trypan blue dye exclusion. Although WI-26 cell death was evident after an 18 h exposure period to CSE and asbestos, it was ;2- to 6-fold less than the levels of DNA-SB detected (Figs. 1 and 4). The type of cell death (e.g., apoptotic or necrotic) was not examined in this study but others have shown that asbestos causes apoptosis that is mediated in part by ROS [30]. It is conceivable that DNA damage in the alveolar epithelium caused by CSE and asbestos could elude proper DNA repair and cell death pathways resulting in the formation of a malignant clone of cells. Considerable evidence, reviewed elsewhere [5], suggest that iron-induced ROS are partially responsible for causing DNA damage after exposure to CSE and asbestos. In this study, we showed that an iron chelator, phytic acid, and catalase, each significantly reduce A549 cell DNA damage and ATP depletion caused by CSE and asbestos. In contrast, neither agent attenuated WI-26 cell DNA damage as well as GSH depletion in both cells. It is possible that the iron bound to the tar components of CSE, such as quinones, are involved in mediating AEC DNA damage. Pryor and coworkers [8,9] demonstrated in a cell-free system that iron chelators, hydroxyl radical scavengers, and catalase each inhibit the formation of DNA nicks and electron spin resonance spectroscopy signals generated by aqueous extracts of cigarette tar. We utilized phytic acid because, just as deferoxamine, it occludes all the reactive coordination sites for iron making it inert, which, subsequently, may protect cells by maintaining iron in the oxidized state [31]. However,
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unlike deferoxamine, phytic acid is a safe, natural food additive that does not scavenge ROS [31]. The protective effects of phytic acid noted in this study are comparable to that provided by deferoxamine against asbestos-induced lipid peroxidation, DNA-SB and cellular toxicity in different target cells as well as DNA damage caused by asbestos and CSE in a cell-free system [5,10]. We previously demonstrated that phytic acid, but not ironloaded phytic acid, attenuates asbestos induced WI-26 cell chromium-51 release and the generation of •OH-like species [11]. These findings suggest that the iron chelating properties of phytic acid are important in protecting AEC exposed to asbestos and CSE. These data also implicate iron-catalyzed ROS in causing synergistic DNA damage and ATP depletion in A549 cells after exposure to CSE and asbestos whereas the mechanisms underlying WI-26 cell DNA damage by both agents remain unclear. The lack of a protective effect with an iron chelator in WI-26 cells does not exclude the possibility that ironinduced ROS are involved. Pryor and Stone [8] suggested that metal ions may be firmly attached with tar components or with the DNA thereby preventing access to the chelator. These investigators proposed that tar radicals intimately associated with the DNA generate • OH, which may not be blocked due to the short diffusion radius of the highly reactive •OH. The selective protective effects of phytic acid and catalase in A549 rather than WI-26 cells may also relate to differences in antioxidant defenses. ATII cells, as compared to ATI cells, are more resistant to an oxidant stress that is in part due to differences in their antioxidant defenses [24]. We found that the GSH levels in WI-26 cells are ;10-fold less than in A549 cells (Fig. 6) but that both cells have comparable catalase activity (D. W. Kamp, 1995, unpublished observation). The reduced levels of GSH may render WI-26 cells particularly sensitive to an oxidant stress. The inability of exogenous catalase to protect WI-26 cells may also be due to hydroquinones and aged catechols, two major constituents of CSE that can each inhibit catalase activity and nick DNA [32]. Pryor and colleagues [8,21,33,34] have shown that gas-phase cigarette smoke contains up to 500 ppm of nitric oxide, which can form peroxynitrite and peroxynitrate esters that subsequently reduce cellular thiols and promote apoptotic cell death in lymphocytes. Although we have not identified the specific agent(s) responsible for the effects observed in this study, one constituent in CSE that may mediate both DNA damage and cell death is H2O2. As mentioned previously, CSE can generate H2O2, which, subsequently, could contribute to the interactive effects between the iron in asbestos through Fenton-type chemistry. The protective effects of catalase and phytic acid noted in this study support this
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hypothesis. Another possibility is iron that can accumulate on the tar in CSE. We noted that CSE alone induced dose-dependent AEC DNA damage. Churg and colleagues [4] suggested that iron-catalyzed ROS also enhance asbestos retention in the lungs because catalase and deferoxamine each reduced cigarette smoke-induced epithelial fiber uptake and translocation in tracheal organ cultures. Collectively, these data suggest that AEC DNA damage caused by iron-induced radical formation is one mechanism that accounts for pulmonary toxicity after exposure to asbestos and cigarette smoke. Despite evidence implicating iron-induced ROS, it should be emphasized that other mechanisms may also have an important role in mediating AEC damage after exposure to cigarette smoke and asbestos. First, radicals derived from nitric oxide in the gas-phase of cigarette smoke may also contribute to lung injury [20,33–35]. Gas-phase cigarette smoke causes dose-dependent DNA-SB formation in cultured human bronchial epithelial cells [35]. Although the pattern of DNA base damage by gas-phase cigarette smoke was consistent with attack by •OH, the most common base damage was the formation of xanthine and hypoxanthine presumably due to deamination of guanine and adenosine, respectively, by reactive nitrogen species [35]. A role for reactive nitrogen species in our model is unclear. However, we have observed that inhibitors of inducible nitric oxide synthase do not attenuate asbestos-induced A549 cell DNA damage over 24 h [36]. Second, benzo[a]pyrene, a major carcinogen in cigarette smoke, can form 6-oxobenzo[a]pyrene free radicals that mediate AEC injury and DNA damage. Asbestos catalyzes the oxidation of 6-hydroxy-benzo-[a]pyrene, a metabolite of benzo[a]pyrene, to the 6-oxobenzo[a]pyrene free radical [37]. Additionally, formation of this radical is not inhibited by deferoxamine and occurs equally well in an oxygen-depleted environment [37]. Finally, aqueous CSE also stimulate c-fos expression, a proto-oncogene involved in cell proliferation and apoptosis after exposure to tumor promoters, oxidant stress, GSH depletion, or DNA damage [38]. Notably, the mechanism seems independent of the production of iron-induced ROS because catalase and an iron chelator failed to block c-fos expression despite the fact that these agents significantly prevented DNA-SB formation [38]. Asbestos and cigarette smoke can reduce GSH levels in various pulmonary cells, which may contribute to the pathogenic effect of each agent [13,14,26,39,40]. Depletion of GSH is due in part to inhibition of enzymes involved in GSH synthesis and to enhanced GSH-dependent conjugation reactions and membrane GSH leakage after exposure to aldehydes in cigarette smoke, such as acrolein, formaldehyde and acetaldehyde [14,39]. In contrast to asbestos, we demonstrated that CSE significantly
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reduce GSH levels in A549 and WI-26 cells but were unable to detect a synergistic interaction (Fig. 5). Our data also suggest that the combination of CSE and asbestos deplete GSH levels by mechanisms independent of iron-induced radical formation because phytic acid and catalase were not protective (Fig. 7). The profound reductions in GSH after AEC were exposed to CSE may have promoted cell death. Although not specifically investigated in this study, aldehydes in cigarette smoke deplete GSH in bronchial epithelial cells by mechanisms that are not coupled to oxidative stress [39]. As mentioned, peroxynitrite can also deplete cellular GSH and contribute to cell death [8,20,33]. ATP depletion is one of the many biochemical events that can occur in cells exposed to ROS and, if severe, can also augment cell death [17,29]. We were unable to detect a synergistic reduction in A549 and WI-26 cell ATP levels after exposure to CSE and asbestos. However, we found that CSE plus asbestos induces DNA-SB formation without necessarily reducing ATP and GSH levels in AEC (Figs. 4, 5, and 6). Therefore, an abnormal clone of cells may originate if either the DNA repair mechanisms or cell death pathways are incomplete. In summary, we have demonstrated that CSE and asbestos can cause DNA-SB formation and cell death in cultured AEC, relevant target cells of both agents. While the interactive effects between CSE and asbestos were primarily additive, synergistic DNA damage and cell death were noted under some conditions. Our results indicate that iron-induced free radicals contribute to A549 cell DNA damage and ATP depletion and that DNA damage can occur without necessarily altering the levels of ATP or GSH. In contrast to A549 cells, DNA damage caused by CSE and asbestos in ATI-like cells (WI-26 and RAEC) was not synergistic nor was phytic acid or catalase protective. We also showed that CSE significantly reduced GSH levels in A549 and WI-26 cells and that phytic acid and catalase did not reduce these effects. We conclude that the mechanism causing alveolar epithelial cell toxicity after exposure to cigarette smoke and asbestos is due in part to DNA damage by iron-induced free radicals. The DNA damaging effects of both agents depend on the cell type exposed and are not necessarily associated with alterations in cellular energy levels or GSH. Although the relevance of our in vitro findings require additional study, Churg and associates [41] demonstrated in rats that amosite asbestos and cigarette smoke induce a brief significant synergistic increase in the DNA labeling index in small airway epithelial cells but not in vascular or mesothelial cells. We speculate that one mechanism that accounts for the enhanced incidence of bronchogenic carcinoma after exposure to cigarette smoke and asbestos is due to iron-
induced ROS that damage DNA in the pulmonary epithelium. Acknowledgements—This work was supported in part by a grant from the Department of Veterans Affairs (Merit Review) and the Asbestos Victims Special Fund Trust, a publicly supported charitable organization. The contents of this article do not necessarily reflect the views of the Trust or the parties that support it. The authors are grateful for the technical assistance provided by Chen Zhang and Elsa Escalara.
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ABBREVIATIONS
AEC—alveolar epithelial cells GSH— glutathione CSE— cigarette smoke extracts • OH— hydroxyl radicals ROS—reactive oxygen species DNA-SB—DNA strand breaks AOE—antioxidant enzymes PBS—phosphate buffered saline H2O2— hydrogen peroxide ATII—alveolar type II DMEM—Dulbecco’s modified Eagle medium RAEC—rat alveolar type II cell cultured for 7–10 d FBS—fetal bovine serum HBSS—Hanks balance salt solution plus calcium, magnesium and 15 mM N-2-hydroxy-ethylpiperazine-N92-ethanesulfonic acid DMSO— dimethylsulfoxide