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Asbestos and cigarette smoke cause increased DNA strand breaks and necrosis in bronchiolar epithelial cells in vivo
Free Radical Biology & Medicine, Vol. 28, No. 8, pp. 1295–1299, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter
PII S0891-5849(00)00211-2
Fast Track Paper ASBESTOS AND CIGARETTE SMOKE CAUSE INCREASED DNA STRAND BREAKS AND NECROSIS IN BRONCHIOLAR EPITHELIAL CELLS IN VIVO MICHAEL JUNG,* WENDELL P. DAVIS,* DOUGLAS J. TAATJES,* ANDREW CHURG,†
and
BROOKE T. MOSSMAN*
*Departments of Pathology, University of Vermont College of Medicine, Burlington, VT, USA; and †University of British Columbia, Vancouver, British Columbia, Canada (Received 10 January 2000; Revised 24 February 2000; Accepted 24 February 2000)
Abstract—Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers. Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species (ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo). We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbers of cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmission electron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestos alone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strand breakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelial cells of the respiratory tract, thus validating in vitro observations from a number of laboratories. © 2000 Elsevier Science Inc.
INTRODUCTION
have not been elucidated in vivo. The objective of work here was to determine whether DNA strand breakage is an in vivo endpoint of injury by cigarette smoke and asbestos to epithelial cells of the respiratory tract. Moreover, we wanted to determine the relationship between patterns of epithelial cell injury to cell proliferation by these agents, alone and in combination, as previously characterized in this rodent model [11]. Male Sprague Dawley rats weighing approximately 250 g were divided into four treatment groups (n ⫽ 4/group): (i) clean air (sham); (ii) amosite asbestos alone (International Union Against Cancer reference sample administered as a single intratracheal instillation of 2.5 mg in 0.5 ml physiologic saline using light halothane anaesthesia at time 0); (iii) cigarette smoke exposure alone; and (iv) cigarette smoke administered for various time periods at 1 h after injection of asbestos (i.e., combination group). Smoke exposures for 1, 2, or 14 d were carried out in a nose-only smoking apparatus as previously described [11,12]. Each rat was exposed daily to the whole smoke of seven commercial nonfilter cigarettes. All animals were killed 24 h after the last smoke exposure by urethane overdose, and the lungs removed and fixed for 24 h by intratracheal inflation with paraformaldehyde to 10 cm of water pressure. The left lung
Cigarette smoking increases the incidence of asbestosassociated lung cancers in heavily exposed worker populations (reviewed in [1], but the mechanisms of injury and cocarcinogenesis by these agents in target cells of the lung are unclear. Work by a number of laboratories shows that asbestos and cigarette smoke generate ROS as well as increases in antioxidant enzymes in tracheobronchial and pulmonary epithelial cells [2– 4]. Cigarette smoke also increases pulmonary retention [5] and uptake of fibers and particles by tracheal epithelial cells through oxidant-dependent mechanisms [6], and ROS may be important in cell injury and transformation by both asbestos and cigarette smoke (reviewed in [2,7]). Although DNA damage and deletions by these agents have been demonstrated biochemically in some experimental models [8,9] and may play a role in alveolar epithelial cell injury in vitro [10], the phenotypic ramifications of asbestos and cigarette smoke on bronchial epithelial cells, the progenitor cell types of bronchogenic carcinomas, Address correspondence to: Dr. B. T. Mossman, University of Vermont College of Medicine, Department of Pathology, Medical Alumni Building, Burlington, VT 05405, USA; Tel: (802) 656-0382; Fax: (802) 656-8892; E-Mail: [email protected]. 1295
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Fig. 1. Demonstration of TUNEL-positive nuclei (brown staining indicated by arrows) in epithelial cells in rat lungs: (A) Sham-exposed rat lung at 1 d at 475 ⫻ magnification. (B) Asbestos-exposed bronchiolar epithelium at 14 d. (C) Bronchiole exposed to asbestos and cigarette smoke at 1 d. (D) Smoke and asbestos-exposed bronchiole at 14 d. (B–D) are 950 ⫻ magnification.
was dehydrated, embedded in paraffin, and sections cut at 5 m thickness. Rats exposed to asbestos, cigarette smoke or the two in combination did not lose weight and appeared to have the same activity patterns as sham rats. DNA strand breaks were evaluated using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method, which detects 3⬘-OH ends of single-stranded DNA. This technique detects both apoptotic and necrotic cells [13]. In studies here, rat lung sections were deparaffinized in xylene, rehydrated through a series of graded alcohol washes (100, 95, 75, and 50%), and rinsed twice for 3 min in phosphate-buffered saline (PBS). Tissue sections were then permeabilized with 0.5% Triton-X in PBS for 5 min followed by two rinses in PBS. To reduce nonspecific binding and deproteinize, sections were washed in 0.2 M HCl for 8 min, followed by two rinses in PBS. To remove endogenous peroxidase activity, sections were treated with 2% H2O2 in ethanol for 5 min and rinsed twice in PBS. In the TUNEL reaction mixture, 10 l/ section of TUNEL enzyme (terminal deoxynucleotidyl transferase in a reaction buffer of 200 mM cacodylic acid, 200 mM KCl, 1mM ethylenediaminetetraacetate (EDTA), 4 mM -mercaptoethanol, 50% glycerine, pH 6.5) (Boehringer Mannheim, Indianapolis, IN, USA) was used in combination with 90 l/section of TUNEL label
(Boehringer Mannheim). As a negative control, TUNEL label (fluorescein-dUTP and unlabeled dUTP in a reaction buffer of 200 mM potassium cacodylate, 25 mM Tris-HCl, 1 mM CaCl2, 0.25 ng/ml bovine serum albumin, pH 6.6) was applied in the absence of enzyme. To prevent evaporation, each section was covered with parafilm and then incubated for 30 min at 37°C, followed by three washes in PBS. Conversion was conducted via application of 75 l antifluorescein antibody (Fab fragment from sheep, conjugated with peroxidase; TUNEL POD; Boehringer Mannheim) per section and incubation for 30 min at 37°C, followed by three washes in PBS. Sections were then stained with diaminobenzidine (DAB)-H2O2 solution for 5 min and rinsed in PBS. Finally, hematoxylin was used as a counterstain, sections were dehydrated through a series of graded alcohols (50, 75, and 100%), and rinsed in xylene. All rinses and washes were carried at room temperature unless otherwise specified. Lung sections were evaluated using an Olympus BX50 upright compound light microscope. Digital images (640 ⫻ 480 pixels) were captured with a microscope-mounted Sony DXC-960/MDLLP modified ccd camera attached to a frame grabber board in a SunSPARC station 5 using the Image Capture module of IMIX Imagist software (Princeton Gamma Tech, Prince-
DNA breaks by asbestos and smoke
ton, NJ) on the workstation. Digital image processing and semi-quantitative analysis was accomplished using a Feature Analysis module. Digital images were stored on 1 gigabyte optical disks. Hard copy photographs were made from the digital images with a Mitsubishi CP1000 color video dye sublimation printer and publication photographs were made with a Fujix Pictrography 3000 video printer. Epithelial cells in distinct membranous (terminal) bronchioles first were evaluated at 400⫻ magnification. For each time point, data were collected for each animal (n ⫽ 4/group) as follows: the number of TUNEL-positive cells per unit length from 5 randomly selected bronchioles on each of two sections/animal were evaluated and expressed as TUNEL- positive cells per length of bronchiolar epithelium. Selected tissues were also examined by TEM to determine if cell death occurred by necrosis or apoptosis. Lungs that were already fixed in 4% phosphate-buffered formaldehyde for 24 h were rinsed 3⫻ (5 mins each) in Millonig’s phosphate buffer, followed by further fixation in half-strength Karnovsky’s fixative (2.5% glutaraldehyde, 60% formaldehyde in Millonig’s buffer) for 2 hrs at 4°C. The tissue was washed in buffer (3 ⫻ 5 min), followed by postfixation in 1% osmium tetroxide in buffer for 30 min at 4°C. Finally, the tissue pieces were dehydrated in a graded series of ethanols, and infiltrated and embedded in Spurr’s epoxy resin. Ultrathin sections were cut with a diamond knife, contrasted with uranyl acetate and lead citrate, and viewed with a JEOL1210 electron microscope operated at 60 kV. In all rat lungs, cells exhibiting DNA strand breaks were predominately observed in the bronchiolar epithelium with little to no TUNEL-positive cells in other compartments of the lung. Sham control animals revealed few TUNEL-positive cells in the intact bronchiolar epithelium (Fig. 1A). However, an occasional superficial or sloughing ciliated epithelial cell labeled intensely. In comparison to sham controls, TUNEL-positive epithelial cells, both basal and superficial, were noted in rat membranous bronchioles exposed to asbestos, cigarette smoke or the two in combination (Figs. 1B–1D). Frequently, TUNEL-positive sloughing epithelial cells were observed in the lumen of the airway. After exposure to asbestos and/or cigarette smoke, some alveolar type II epithelial cells occasionally stained positively by the TUNEL method. These patterns of injury are consistent with observations in human lungs where both cigarette smoke and asbestos exposures have been shown to produce inflammatory and fibrotic changes in membranous and respiratory bronchioles. To quantitate numbers of TUNEL-positive cells induced by agents over time, computer-assisted image analysis was employed. In comparison to sham controls, numbers of TUNEL-positive epithelial cells in distinct
1297
Fig. 2. Quantitation of DNA breaks in bronchiolar epithelial cells from rat lung sections. All membranous bronchioles were evaluated on each of 2 lung sections from each animal (n ⫽ 4 rats/group/time point). Mean ⫾ SEM of each group. *p ⫽ .054.
membranous bronchioles of animals were increased approximately 4-fold in asbestos, smoke and smoke plus asbestos groups at day 1 (Fig. 2). At 14 d, striking increases were observed in the group exposed to asbestos alone, and additive effects were seen in smoke and asbestos-exposed rat lungs (p ⫽ .054). TEM verified that TUNEL-positive cells reflected primarily necrotic death of bronchiolar epithelial cells in asbestos and smokeexposed lungs (Fig. 3). These cells exhibited rupture of nuclear and organelle membranes and a swollen cytoplasm with loss of basophilia. Necrotic epithelial cells often appeared in the lumen in the presence of neutrophils or alveolar macrophages containing asbestos fibers (Fig. 4). Cell death is multifaceted and may occur through distinct patterns which are most often classified as necrosis or apoptosis [13,14]. Necrosis is defined as death of cells through external damage which may destroy the integrity of the plasma membrane. Necrotic debris elicits
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Fig. 3. Transmission electron micrograph (TEM) showing necrotic vacuolated epithelial cell at 14 days after exposure to asbestos and cigarette smoke. Magnification ⫽ 2565 ⫻.
inflammation and infiltration of phagocytic cells, consistent with our observations here and inflammatory changes in rodent inhalation models of asbestos-induced lung disease (reviewed in [7]). In contrast, apoptosis or programmed cell death is mechanistically distinct, occurring with the formation of characteristic apoptotic bodies
in a stereotyped sequence of events. Apoptosis may occur spontaneously during normal embryonic development or in response to chemotherapeutic or other agents [15]. Both H2O2 and cigarette smoke cause apoptosis at low concentrations and necrosis at high concentrations in cells in vitro [16,17]. Moreover, exposure to asbestos in vitro yields apoptosis in pleural mesothelial cells which is ameliorated by antioxidants and inhibitors of mitogenactivated protein kinases [18,19]. However, studies using tracheal epithelial organ cultures have demonstrated that necrosis and exfoliation of superficial cells also occur after exposure to asbestos [20,21], and recent studies show additive or synergistic effects of asbestos and smoke on necrotic cell death (as assessed by 51Cr release) and DNA strand breaks (as measured by alkaline unwinding and ethidium bromide fluorescence) in cultured alveolar epithelial cells [10]. Whereas these and other studies provide in vitro evidence that asbestos and cigarette smoke are damaging and genotoxic to cells, data here show that DNA strand breaks occur in lungs after exposure to these agents in vivo. Moreover, this damage culminates in necrotic cell death. Elucidating the type of cell death by asbestos and cigarette smoke or other types of oxidant injury in lung is important in considering that cytoprotective approaches may have differential effects on either necrosis or apoptosis. For example, overexpression of heat shock proteins (HSP70, HSP27), protects against necrosis, but not apoptosis induced by cigarette smoke or H2O2 [16,17]. Necrotic cell injury by asbestos and cigarette smoke may be triggered by DNA damage and can trigger or occur simultaneously with compensatory hyperplasia [20 –22]. Previously, Sekhon et al. [11] documented changes in cell proliferation in the lungs of the same animals examined here using 5⬘bromodeoxyuridine (BrdU) as an indication of increased DNA synthesis. If results here and the studies by Sekhon and colleagues are examined comparatively, significant increases in both BrdU- and TUNEL-positive bronchiolar epithelial cells are signatures of brief exposures to asbestos and cigarette smoke. These alterations may reflect initial sites of deposition of components of cigarette smoke and asbestos fibers which are translocated peripherally in the lung over time.
CONCLUSION
Fig. 4. Transmission electron micrograph (TEM) of asbestos and smoke-exposed lung at 14 d indicating sloughed necrotic epithelial cell (arrow) accompanied by infiltration of neutrophils (N) and alveolar macrophages (M) containing asbestos fibers (arrowhead). Magnification ⫽ 4940 ⫻.
We show here that DNA strand breaks and necrosis are early molecular and phenotypic changes in bronchiolar epithelial cells after exposure to asbestos and cigarette smoke in vivo. The observation that these endpoints of epithelial injury by asbestos or smoking occurs in lungs of rodents substantiates in vitro experiments by us
DNA breaks by asbestos and smoke
and others suggesting that these lung carcinogens damage DNA. Acknowledgements — We thank Dr. Pamela Vacek, Department of Biostatistics, for statistical analyses and Laurie Sabens for preparation of the manuscript. Dr. John E. Craighead provided valuable assistance in examining histopathology. Supported by grants #ES06499 and # ES09213 from National Institute of Environmental Health Sciences and grant #HL39469 from National Heart, Lung, and Blood Institute to B.T.M. and grants from the Medical Research Council of Canada to A.C.
REFERENCES [1] Mossman, B. T.; Gee, J. B. L. Medical progress. Asbestos-related diseases. N. Engl. J. Med. 320:1721–1730; 1989. [2] Kamp, D. W.; Graceffa, P.; Pryor, W. A.; Weitzman, S. A. The role of free radicals in asbestos-associated diseases. Free Radic. Biol. Med. 12:293–315; 1992. [3] Weitzman, S. A.; Graceffa, P. Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide. Arch. Biochem. Biophys. 228:373–376; 1984. [4] Pryor, W. A.; Stone, K. Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrate and peroxynitrite. Ann. NY Acad. Sci. 686:12–27; 1993. [5] Cohen, D.; Arai, S. F.; Brain, J. D. Smoking impairs long-term clearance from the lung. Science 204:514 –516; 1979. [6] Churg, A.; Hobson, J.; Berean, K.; Wright, J. Scavengers of active oxygen species prevent cigarette smoke-induced asbestos fiber penetration in rat tracheal explants. Am. J. Pathol. 135:599 – 603; 1989. [7] Mossman, B. T.; Churg, A. State-of-the-Art: mechanisms in the pathogenesis of asbestosis and silicosis. Am. J. Respir. Crit. Care Med. 157:1–15; 1998. [8] Jalili, T.; Murthy, G. G.; Schiestl, R. H. Cigarette smoke induces DNA deletions in the mouse embryo. Cancer Res. 58:2633–2638; 1998. [9] Jackson, J. H.; Schraufstatter, I. U.; Hyslop, P. A.; Vosbeck, K.; Sauerhber, R.; Weitzman, S. A.; Cochrane, C. G. Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke. J. Clin. Invest. 80:1090 –1095; 1987.
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[10] Kamp, D. W.; Greenberger, M. J.; Sbalchierro, J. S.; Preusen, S. E.; Weitzman, S. A. Cigarette smoke augments asbestosinduced alveolar epithelial cell injury: role of free radicals. Free Radic. Biol. Med. 25:728 –739; 1998. [11] Sekhon, H.; Wright, J.; Churg, A. Effects of cigarette smoke and asbestos on airway, vascular, and mesothelial cell proliferation. Int. J. Exp. Pathol. 76:411– 418; 1995. [12] Sekhon, H.; Wright, J. L.; Churg, A. Cigarette smoke causes rapid cell proliferation in small airways and associated pulmonary arteries. Am. J. Physiol. 268:L557–L563; 1994. [13] Willingham, M. C. Cytochemical methods for the detection of apoptosis. J. Histochem. Cytochem. 47:1101–1109; 1999. [14] Majno, G.; Joris, I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146:3–15; 1995. [15] Wyllie, A. H.; Kerr, J. F. R.; Currie, A. R. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68:251–306; 1980. [16] Vayssier, M.; Banzet, N.; Francois, D.; Bellmann, K.; Polla, B. S. Tobacco smoke induces both apoptosis and necrosis in mammalian cells: differential effects of HSP70. Am. J. Physiol. 275: L771–L779; 1998. [17] Guenal, I.; Sidoti-de Fraisse, C.; Gaumer, S.; Mignotte, B. Bcl-2 and hsp27 act at different levels to suppress programmed cell death. Oncogene 15:347–360; 1997. [18] Jimenez, L. A.; Zanella, C.; Fung, H.; Janssen, Y. M. W.; Vacek, P.; Charland, C.; Goldberg, J.; Mossman, B. T. Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2. Am. J. Physiol. 273:L1029 –L1035; 1997. [19] Broaddus, V. C.; Yang, L.; Scavo, L. M.; Ernst, J. D.; Boylan, A. M. Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species. J. Clin. Invest. 98:2050 –2059; 1996. [20] Woodworth, C. D.; Mossman, B. T.; Craighead, J. E. Induction of squamous metaplasia in organ cultures of hamster trachea by naturally occurring and synthetic cultures. Cancer Res. 43:4906 – 4913; 1983. [21] Mossman, B. T.; Craighead, J. E.; MacPherson, B. V. Asbestosinduced epithelial changes in organ cultures of hamster trachea: inhibition by retinyl methyl ether. Science 207:311–313; 1980. [22] Mossman, B. T.; Eastman, A.; Bresnick, E. Asbestos and benzo[a]pyrene act synergistically to induce squamous metaplasia and incorporation of [3H]thymidine in hamster tracheal epithelium. Carcinogenesis 5:1401–1404; 1984.
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Fast Track Paper ASBESTOS AND CIGARETTE SMOKE CAUSE INCREASED DNA STRAND BREAKS AND NECROSIS IN BRONCHIOLAR EPITHELIAL CELLS IN VIVO MICHAEL JUNG,* WENDELL P. DAVIS,* DOUGLAS J. TAATJES,* ANDREW CHURG,†
and
BROOKE T. MOSSMAN*
*Departments of Pathology, University of Vermont College of Medicine, Burlington, VT, USA; and †University of British Columbia, Vancouver, British Columbia, Canada (Received 10 January 2000; Revised 24 February 2000; Accepted 24 February 2000)
Abstract—Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers. Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species (ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo). We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbers of cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmission electron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestos alone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strand breakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelial cells of the respiratory tract, thus validating in vitro observations from a number of laboratories. © 2000 Elsevier Science Inc.
INTRODUCTION
have not been elucidated in vivo. The objective of work here was to determine whether DNA strand breakage is an in vivo endpoint of injury by cigarette smoke and asbestos to epithelial cells of the respiratory tract. Moreover, we wanted to determine the relationship between patterns of epithelial cell injury to cell proliferation by these agents, alone and in combination, as previously characterized in this rodent model [11]. Male Sprague Dawley rats weighing approximately 250 g were divided into four treatment groups (n ⫽ 4/group): (i) clean air (sham); (ii) amosite asbestos alone (International Union Against Cancer reference sample administered as a single intratracheal instillation of 2.5 mg in 0.5 ml physiologic saline using light halothane anaesthesia at time 0); (iii) cigarette smoke exposure alone; and (iv) cigarette smoke administered for various time periods at 1 h after injection of asbestos (i.e., combination group). Smoke exposures for 1, 2, or 14 d were carried out in a nose-only smoking apparatus as previously described [11,12]. Each rat was exposed daily to the whole smoke of seven commercial nonfilter cigarettes. All animals were killed 24 h after the last smoke exposure by urethane overdose, and the lungs removed and fixed for 24 h by intratracheal inflation with paraformaldehyde to 10 cm of water pressure. The left lung
Cigarette smoking increases the incidence of asbestosassociated lung cancers in heavily exposed worker populations (reviewed in [1], but the mechanisms of injury and cocarcinogenesis by these agents in target cells of the lung are unclear. Work by a number of laboratories shows that asbestos and cigarette smoke generate ROS as well as increases in antioxidant enzymes in tracheobronchial and pulmonary epithelial cells [2– 4]. Cigarette smoke also increases pulmonary retention [5] and uptake of fibers and particles by tracheal epithelial cells through oxidant-dependent mechanisms [6], and ROS may be important in cell injury and transformation by both asbestos and cigarette smoke (reviewed in [2,7]). Although DNA damage and deletions by these agents have been demonstrated biochemically in some experimental models [8,9] and may play a role in alveolar epithelial cell injury in vitro [10], the phenotypic ramifications of asbestos and cigarette smoke on bronchial epithelial cells, the progenitor cell types of bronchogenic carcinomas, Address correspondence to: Dr. B. T. Mossman, University of Vermont College of Medicine, Department of Pathology, Medical Alumni Building, Burlington, VT 05405, USA; Tel: (802) 656-0382; Fax: (802) 656-8892; E-Mail: [email protected]. 1295
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Fig. 1. Demonstration of TUNEL-positive nuclei (brown staining indicated by arrows) in epithelial cells in rat lungs: (A) Sham-exposed rat lung at 1 d at 475 ⫻ magnification. (B) Asbestos-exposed bronchiolar epithelium at 14 d. (C) Bronchiole exposed to asbestos and cigarette smoke at 1 d. (D) Smoke and asbestos-exposed bronchiole at 14 d. (B–D) are 950 ⫻ magnification.
was dehydrated, embedded in paraffin, and sections cut at 5 m thickness. Rats exposed to asbestos, cigarette smoke or the two in combination did not lose weight and appeared to have the same activity patterns as sham rats. DNA strand breaks were evaluated using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method, which detects 3⬘-OH ends of single-stranded DNA. This technique detects both apoptotic and necrotic cells [13]. In studies here, rat lung sections were deparaffinized in xylene, rehydrated through a series of graded alcohol washes (100, 95, 75, and 50%), and rinsed twice for 3 min in phosphate-buffered saline (PBS). Tissue sections were then permeabilized with 0.5% Triton-X in PBS for 5 min followed by two rinses in PBS. To reduce nonspecific binding and deproteinize, sections were washed in 0.2 M HCl for 8 min, followed by two rinses in PBS. To remove endogenous peroxidase activity, sections were treated with 2% H2O2 in ethanol for 5 min and rinsed twice in PBS. In the TUNEL reaction mixture, 10 l/ section of TUNEL enzyme (terminal deoxynucleotidyl transferase in a reaction buffer of 200 mM cacodylic acid, 200 mM KCl, 1mM ethylenediaminetetraacetate (EDTA), 4 mM -mercaptoethanol, 50% glycerine, pH 6.5) (Boehringer Mannheim, Indianapolis, IN, USA) was used in combination with 90 l/section of TUNEL label
(Boehringer Mannheim). As a negative control, TUNEL label (fluorescein-dUTP and unlabeled dUTP in a reaction buffer of 200 mM potassium cacodylate, 25 mM Tris-HCl, 1 mM CaCl2, 0.25 ng/ml bovine serum albumin, pH 6.6) was applied in the absence of enzyme. To prevent evaporation, each section was covered with parafilm and then incubated for 30 min at 37°C, followed by three washes in PBS. Conversion was conducted via application of 75 l antifluorescein antibody (Fab fragment from sheep, conjugated with peroxidase; TUNEL POD; Boehringer Mannheim) per section and incubation for 30 min at 37°C, followed by three washes in PBS. Sections were then stained with diaminobenzidine (DAB)-H2O2 solution for 5 min and rinsed in PBS. Finally, hematoxylin was used as a counterstain, sections were dehydrated through a series of graded alcohols (50, 75, and 100%), and rinsed in xylene. All rinses and washes were carried at room temperature unless otherwise specified. Lung sections were evaluated using an Olympus BX50 upright compound light microscope. Digital images (640 ⫻ 480 pixels) were captured with a microscope-mounted Sony DXC-960/MDLLP modified ccd camera attached to a frame grabber board in a SunSPARC station 5 using the Image Capture module of IMIX Imagist software (Princeton Gamma Tech, Prince-
DNA breaks by asbestos and smoke
ton, NJ) on the workstation. Digital image processing and semi-quantitative analysis was accomplished using a Feature Analysis module. Digital images were stored on 1 gigabyte optical disks. Hard copy photographs were made from the digital images with a Mitsubishi CP1000 color video dye sublimation printer and publication photographs were made with a Fujix Pictrography 3000 video printer. Epithelial cells in distinct membranous (terminal) bronchioles first were evaluated at 400⫻ magnification. For each time point, data were collected for each animal (n ⫽ 4/group) as follows: the number of TUNEL-positive cells per unit length from 5 randomly selected bronchioles on each of two sections/animal were evaluated and expressed as TUNEL- positive cells per length of bronchiolar epithelium. Selected tissues were also examined by TEM to determine if cell death occurred by necrosis or apoptosis. Lungs that were already fixed in 4% phosphate-buffered formaldehyde for 24 h were rinsed 3⫻ (5 mins each) in Millonig’s phosphate buffer, followed by further fixation in half-strength Karnovsky’s fixative (2.5% glutaraldehyde, 60% formaldehyde in Millonig’s buffer) for 2 hrs at 4°C. The tissue was washed in buffer (3 ⫻ 5 min), followed by postfixation in 1% osmium tetroxide in buffer for 30 min at 4°C. Finally, the tissue pieces were dehydrated in a graded series of ethanols, and infiltrated and embedded in Spurr’s epoxy resin. Ultrathin sections were cut with a diamond knife, contrasted with uranyl acetate and lead citrate, and viewed with a JEOL1210 electron microscope operated at 60 kV. In all rat lungs, cells exhibiting DNA strand breaks were predominately observed in the bronchiolar epithelium with little to no TUNEL-positive cells in other compartments of the lung. Sham control animals revealed few TUNEL-positive cells in the intact bronchiolar epithelium (Fig. 1A). However, an occasional superficial or sloughing ciliated epithelial cell labeled intensely. In comparison to sham controls, TUNEL-positive epithelial cells, both basal and superficial, were noted in rat membranous bronchioles exposed to asbestos, cigarette smoke or the two in combination (Figs. 1B–1D). Frequently, TUNEL-positive sloughing epithelial cells were observed in the lumen of the airway. After exposure to asbestos and/or cigarette smoke, some alveolar type II epithelial cells occasionally stained positively by the TUNEL method. These patterns of injury are consistent with observations in human lungs where both cigarette smoke and asbestos exposures have been shown to produce inflammatory and fibrotic changes in membranous and respiratory bronchioles. To quantitate numbers of TUNEL-positive cells induced by agents over time, computer-assisted image analysis was employed. In comparison to sham controls, numbers of TUNEL-positive epithelial cells in distinct
1297
Fig. 2. Quantitation of DNA breaks in bronchiolar epithelial cells from rat lung sections. All membranous bronchioles were evaluated on each of 2 lung sections from each animal (n ⫽ 4 rats/group/time point). Mean ⫾ SEM of each group. *p ⫽ .054.
membranous bronchioles of animals were increased approximately 4-fold in asbestos, smoke and smoke plus asbestos groups at day 1 (Fig. 2). At 14 d, striking increases were observed in the group exposed to asbestos alone, and additive effects were seen in smoke and asbestos-exposed rat lungs (p ⫽ .054). TEM verified that TUNEL-positive cells reflected primarily necrotic death of bronchiolar epithelial cells in asbestos and smokeexposed lungs (Fig. 3). These cells exhibited rupture of nuclear and organelle membranes and a swollen cytoplasm with loss of basophilia. Necrotic epithelial cells often appeared in the lumen in the presence of neutrophils or alveolar macrophages containing asbestos fibers (Fig. 4). Cell death is multifaceted and may occur through distinct patterns which are most often classified as necrosis or apoptosis [13,14]. Necrosis is defined as death of cells through external damage which may destroy the integrity of the plasma membrane. Necrotic debris elicits
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Fig. 3. Transmission electron micrograph (TEM) showing necrotic vacuolated epithelial cell at 14 days after exposure to asbestos and cigarette smoke. Magnification ⫽ 2565 ⫻.
inflammation and infiltration of phagocytic cells, consistent with our observations here and inflammatory changes in rodent inhalation models of asbestos-induced lung disease (reviewed in [7]). In contrast, apoptosis or programmed cell death is mechanistically distinct, occurring with the formation of characteristic apoptotic bodies
in a stereotyped sequence of events. Apoptosis may occur spontaneously during normal embryonic development or in response to chemotherapeutic or other agents [15]. Both H2O2 and cigarette smoke cause apoptosis at low concentrations and necrosis at high concentrations in cells in vitro [16,17]. Moreover, exposure to asbestos in vitro yields apoptosis in pleural mesothelial cells which is ameliorated by antioxidants and inhibitors of mitogenactivated protein kinases [18,19]. However, studies using tracheal epithelial organ cultures have demonstrated that necrosis and exfoliation of superficial cells also occur after exposure to asbestos [20,21], and recent studies show additive or synergistic effects of asbestos and smoke on necrotic cell death (as assessed by 51Cr release) and DNA strand breaks (as measured by alkaline unwinding and ethidium bromide fluorescence) in cultured alveolar epithelial cells [10]. Whereas these and other studies provide in vitro evidence that asbestos and cigarette smoke are damaging and genotoxic to cells, data here show that DNA strand breaks occur in lungs after exposure to these agents in vivo. Moreover, this damage culminates in necrotic cell death. Elucidating the type of cell death by asbestos and cigarette smoke or other types of oxidant injury in lung is important in considering that cytoprotective approaches may have differential effects on either necrosis or apoptosis. For example, overexpression of heat shock proteins (HSP70, HSP27), protects against necrosis, but not apoptosis induced by cigarette smoke or H2O2 [16,17]. Necrotic cell injury by asbestos and cigarette smoke may be triggered by DNA damage and can trigger or occur simultaneously with compensatory hyperplasia [20 –22]. Previously, Sekhon et al. [11] documented changes in cell proliferation in the lungs of the same animals examined here using 5⬘bromodeoxyuridine (BrdU) as an indication of increased DNA synthesis. If results here and the studies by Sekhon and colleagues are examined comparatively, significant increases in both BrdU- and TUNEL-positive bronchiolar epithelial cells are signatures of brief exposures to asbestos and cigarette smoke. These alterations may reflect initial sites of deposition of components of cigarette smoke and asbestos fibers which are translocated peripherally in the lung over time.
CONCLUSION
Fig. 4. Transmission electron micrograph (TEM) of asbestos and smoke-exposed lung at 14 d indicating sloughed necrotic epithelial cell (arrow) accompanied by infiltration of neutrophils (N) and alveolar macrophages (M) containing asbestos fibers (arrowhead). Magnification ⫽ 4940 ⫻.
We show here that DNA strand breaks and necrosis are early molecular and phenotypic changes in bronchiolar epithelial cells after exposure to asbestos and cigarette smoke in vivo. The observation that these endpoints of epithelial injury by asbestos or smoking occurs in lungs of rodents substantiates in vitro experiments by us
DNA breaks by asbestos and smoke
and others suggesting that these lung carcinogens damage DNA. Acknowledgements — We thank Dr. Pamela Vacek, Department of Biostatistics, for statistical analyses and Laurie Sabens for preparation of the manuscript. Dr. John E. Craighead provided valuable assistance in examining histopathology. Supported by grants #ES06499 and # ES09213 from National Institute of Environmental Health Sciences and grant #HL39469 from National Heart, Lung, and Blood Institute to B.T.M. and grants from the Medical Research Council of Canada to A.C.
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