ENVIRONMENTAL RESEARCH 55, 145--156 (1991)
Dehydroepiandrosterone Inhibits the Spontaneous Release of Superoxide Radical by Alveolar Macrophages in Vitro in Asbestosis W I L L I A M N . ROM AND TIMOTHY HARKIN
Division of Pulmonary and Critical Care Medicine, Departments o f Medicine and Environmental Medicine, and Chest Service, Bellevue Hospital, N e w York University Medical Center, 550 First Avenue, New York, New York 10016 Received December 12, 1989
Asbestosis is characterized by an alveolar macrophage alveolitis with injury and fibrosis of the lower respiratory tract. Alveolar macrophages recovered by bronchoalveolar lavage spontaneously release exaggerated amounts of oxidants including superoxide anion and hydrogen peroxide that may mediate alveolar epithelial cell injury. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that inhibits glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate shunt necessary for NADPH generation and superoxide anion formation. In this regard, we hypothesized that DHEA may reduce asbestos-induced oxidant release. DHEA added in vitro to alveolar macrophages lavaged from 11 nonsmoking asbestos workers significantly reduced superoxide anion release. DHEA was measured in bronchoalveolar lavage and found to be similar to serum concentrations. DHEA is an antioxidant and potential anticarcinogenic agent that may have a therapeutic role in reducing the increased oxidant burden in asbestos-induced alveolitis of the lower respiratory tract. © 1991 AcademicPress,Inc.
INTRODUCTION
Asbestosis is characterized by an alveolar macrophage alveolitis with activated inflammatory cells spontaneously releasing oxidants capable of injuring alveolar epithelial cells (Rom et al., 1987; Craighead et al., 1982; Rom et al., 1991; Cantin et al., 1989a; Jaurand et al., 1980). Previous investigations using bronchoalveolar lavage in individuals with asbestosis and in animal models have demonstrated that alveolar macrophages have a morphological appearance of activation (Rom et al., 1987; Takemura et al., 1989) and spontaneously release significant amounts of superoxide anion and hydrogen peroxide (Rom et al., 1987; Cantin et al., 1988; Brgin et al., 1983). In vitro exposure to asbestos can stimulate the oxidative metabolism of neutrophils and macrophages (Donaldson et al., 1985; Case et al., 1986). Cigarette smoke can markedly enhance lung inflammatory cell oxidant release. Phagocyte-derived oxidants play an essential role in killing inhaled microorganisms, but can also prove deleterious by inactivating extracellular proteins such as a-l-antitrypsin, inducing lipid peroxidation in cell membranes, causing cytogenetic injury, and mediating cell death (Cantin et al., 1989a; Cantin and Crystal, 1985; Goodglick et al., 1989). Animal models of asbestosis reproduce the interstitial fibrosis seen in humans and antioxidant approaches to therapy can inhibit lung injury, inflammation, and interstitial pulmonary fibrosis (Mossman et al., 1990). Asbestos fibers can translocate across alveolar epithelial cells propelled 145 0013-9351/91 $3.00 Copyright@ 1991by AcademicPress, Inc. All fightsof reproductionin any formreserved.
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by intracellular actin fibrils (Brody et al., 1983). One of the more dramatic events in asbestosis is the proliferation and enlargement of the cuboidal type II alveolar epithelial cells with the loss of the flat, lining type I cells (Craighead et al., 1982). Alveolar epithelial cells in mice 2 days after exposure to crocidolite asbestos increase their DNA synthesis to 10% from 0.3% prior to exposure (Brody and Overby, 1989). There is a 60% increase in the number of alveolar type II epithelial cells and a 90% increase in their cell volume in rat models of asbestosis after 3 months of daily chrysotile exposure (Barry et al., 1983). Asbestos may also release hydroxyl radical in its interaction with cells to cause DNA strand breaks that may be important events in carcinogenesis (Jackson et al., 1987). As measured by fluorescent microscopy, DNA strand breaks increased from 4.3% after crocidolite exposure, and 9.8% after cigarette smoke exposure, to 78 --- 12% after the combined exposure. Using electron paramagnetic resonance, hydroxyl radical was detected and DNA damage was blocked by oxidant scavengers such as mannitol, catalase, iron chelators, and dimethyl sulfoxide. Oxidants released by asbestos-exposed cells in vivo may be an important mechanism in the cell injury of asbestosis, and function as a possible step in carcinogenesis. Thus, it would be rational to identify potential therapeutic strategies to reduce the oxidant burden of the lower respiratory tract following chronic asbestos exposure. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that acts as a noncompetitive inhibitor limiting substrates for the membranebound flavoprotein NADPH oxidase that generates superoxide radical (Marks and Banks, 1960; Whitcomb and Schwartz, 1985). In this regard, DHEA was evaluated as a potential agent to reduce spontaneous oxidant release from inflammatory cells recovered from the lower respiratory tract of nonsmoking individuals with asbestosis. METHODS Study population. There were 11 individuals who had chronic occupational exposure to asbestos for 33 +- 2 years as asbestos insulators, boilermakers, and pipefitters. All were lifelong nonsmokers. All had PA chest X rays t>% according to the 1980 International Classification of Radiographs of the Pneumoconioses and all had pleural changes. Ten of 11 had rales and all had dyspnea on exertion after climbing two flights of stairs. No subject was taking medications. Pulmonary function tests were performed according to ATS recommendations. Bronchoalveolar lavage was performed with a flexible fiberoptic brochoscope as described using a total of 300 ml of normal saline in 5,20 ml aliquots for each of three sites lavaged (Hunninghake et al., 1979). A total cell count was done in a hematocytometer, and cell differentials performed on cytocentrifuge slides stained with Diff-Quick (American Scientific, Edison, N J) and 500 cells counted. Viability was determined by trypan blue exclusion, and in all cases, recovered cells were >85% viable. Measurement o f oxidants. The spontaneous release of oxidants by the recovered alveolar macrophages was evaluated by incubating the macrophages in Hank's Balanced Salt Solution without phenol red at a concentration of 106/ml in Falcon 24-well plates (Becton-Dickinson, Mountain View, CA). Superoxide an-
DHEA [N ASBESTOSIS
147
ion release into the supernatant was measured by determining the reduction of 80 ~M ferricytochrome c (Sigma, St. Louis, MO) by 10 6 cells per hour at 550 nm using a spectrophotometer (Pick and Mizel, 1981). Units were expressed as the amount of cytochrome c reduced, calculated using a reduced-oxidized extinction coefficient of 21.1 mM- 1 c m - l by 106 cells per hour (Massey, 1959). Hydrogen peroxide release was measured by the change in absorbance at 610 nm by 10 6 cells per hour using the phenol red-horseradish peroxidase method (Pick and Keisari, 1980). Superoxide dismutase (0.5 mg/ml, Sigma) was added to reduce superoxide anion release, and catalase (4300 units/ml, Sigma) was added to reduce hydrogen peroxide release. DHEA (Sigma) was dissolved in ethanol and the various concentrations added in a volume of 3 pJ with an ethanol control. Ethanol vehicle as a control and DHEA in vehicle were added at time 0 and the incubation period was 1 hr. All experiments were performed in triplicate. Measurement of DHEA. The lavage fluid recovered from eight additional individuals (one normal, two asbestosis, five other lung disease including four interstitial lung disease) was examined by radioimmunoassay (RIA) for endogenous concentrations of DHEA, using the DHEA test set (No. DS-2100, Wien Laboratories, Succasunna, N J). Epithelial lining fluid (ELF) is diluted approximately 100 times by the technique of BAL (Rennard et al., 1986); therefore the protocol for the RIA was modified by extracting DHEA from a 20-ml aliquot of BAL fluid, which is 100 times the volume of serum the manufacturer recommends for extraction. The modification did not reduce the efficiency of extraction of DHEA, as recovery of a known amount of DHEA using the modified protocol was >99%. Statistics. All data are expressed as the mean _+ standard error of the mean. Comparisons between asbestosis with and without vehicle plus DHEA were made with the paired Student t test. The level of significance accepted was 0.05. RESULTS The nonsmoking asbestos workers with asbestosis had restrictive impairment on pulmonary function tests (Table I), pleural and parenchymal opacities on their chest radiographs, dyspnea upon climbing two flights of stairs, and 10/11 had rales on auscultation of their chest. The asbestos workers had increased cells recovered by bronchoalveolar lavage (asbestosis 380 - 56 x 103 cells/ml, normal
Dehydroepiandrosterone Inhibits the Spontaneous Release of Superoxide Radical by Alveolar Macrophages in Vitro in Asbestosis W I L L I A M N . ROM AND TIMOTHY HARKIN
Division of Pulmonary and Critical Care Medicine, Departments o f Medicine and Environmental Medicine, and Chest Service, Bellevue Hospital, N e w York University Medical Center, 550 First Avenue, New York, New York 10016 Received December 12, 1989
Asbestosis is characterized by an alveolar macrophage alveolitis with injury and fibrosis of the lower respiratory tract. Alveolar macrophages recovered by bronchoalveolar lavage spontaneously release exaggerated amounts of oxidants including superoxide anion and hydrogen peroxide that may mediate alveolar epithelial cell injury. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that inhibits glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate shunt necessary for NADPH generation and superoxide anion formation. In this regard, we hypothesized that DHEA may reduce asbestos-induced oxidant release. DHEA added in vitro to alveolar macrophages lavaged from 11 nonsmoking asbestos workers significantly reduced superoxide anion release. DHEA was measured in bronchoalveolar lavage and found to be similar to serum concentrations. DHEA is an antioxidant and potential anticarcinogenic agent that may have a therapeutic role in reducing the increased oxidant burden in asbestos-induced alveolitis of the lower respiratory tract. © 1991 AcademicPress,Inc.
INTRODUCTION
Asbestosis is characterized by an alveolar macrophage alveolitis with activated inflammatory cells spontaneously releasing oxidants capable of injuring alveolar epithelial cells (Rom et al., 1987; Craighead et al., 1982; Rom et al., 1991; Cantin et al., 1989a; Jaurand et al., 1980). Previous investigations using bronchoalveolar lavage in individuals with asbestosis and in animal models have demonstrated that alveolar macrophages have a morphological appearance of activation (Rom et al., 1987; Takemura et al., 1989) and spontaneously release significant amounts of superoxide anion and hydrogen peroxide (Rom et al., 1987; Cantin et al., 1988; Brgin et al., 1983). In vitro exposure to asbestos can stimulate the oxidative metabolism of neutrophils and macrophages (Donaldson et al., 1985; Case et al., 1986). Cigarette smoke can markedly enhance lung inflammatory cell oxidant release. Phagocyte-derived oxidants play an essential role in killing inhaled microorganisms, but can also prove deleterious by inactivating extracellular proteins such as a-l-antitrypsin, inducing lipid peroxidation in cell membranes, causing cytogenetic injury, and mediating cell death (Cantin et al., 1989a; Cantin and Crystal, 1985; Goodglick et al., 1989). Animal models of asbestosis reproduce the interstitial fibrosis seen in humans and antioxidant approaches to therapy can inhibit lung injury, inflammation, and interstitial pulmonary fibrosis (Mossman et al., 1990). Asbestos fibers can translocate across alveolar epithelial cells propelled 145 0013-9351/91 $3.00 Copyright@ 1991by AcademicPress, Inc. All fightsof reproductionin any formreserved.
146
ROM A N D H A R K I N
by intracellular actin fibrils (Brody et al., 1983). One of the more dramatic events in asbestosis is the proliferation and enlargement of the cuboidal type II alveolar epithelial cells with the loss of the flat, lining type I cells (Craighead et al., 1982). Alveolar epithelial cells in mice 2 days after exposure to crocidolite asbestos increase their DNA synthesis to 10% from 0.3% prior to exposure (Brody and Overby, 1989). There is a 60% increase in the number of alveolar type II epithelial cells and a 90% increase in their cell volume in rat models of asbestosis after 3 months of daily chrysotile exposure (Barry et al., 1983). Asbestos may also release hydroxyl radical in its interaction with cells to cause DNA strand breaks that may be important events in carcinogenesis (Jackson et al., 1987). As measured by fluorescent microscopy, DNA strand breaks increased from 4.3% after crocidolite exposure, and 9.8% after cigarette smoke exposure, to 78 --- 12% after the combined exposure. Using electron paramagnetic resonance, hydroxyl radical was detected and DNA damage was blocked by oxidant scavengers such as mannitol, catalase, iron chelators, and dimethyl sulfoxide. Oxidants released by asbestos-exposed cells in vivo may be an important mechanism in the cell injury of asbestosis, and function as a possible step in carcinogenesis. Thus, it would be rational to identify potential therapeutic strategies to reduce the oxidant burden of the lower respiratory tract following chronic asbestos exposure. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that acts as a noncompetitive inhibitor limiting substrates for the membranebound flavoprotein NADPH oxidase that generates superoxide radical (Marks and Banks, 1960; Whitcomb and Schwartz, 1985). In this regard, DHEA was evaluated as a potential agent to reduce spontaneous oxidant release from inflammatory cells recovered from the lower respiratory tract of nonsmoking individuals with asbestosis. METHODS Study population. There were 11 individuals who had chronic occupational exposure to asbestos for 33 +- 2 years as asbestos insulators, boilermakers, and pipefitters. All were lifelong nonsmokers. All had PA chest X rays t>% according to the 1980 International Classification of Radiographs of the Pneumoconioses and all had pleural changes. Ten of 11 had rales and all had dyspnea on exertion after climbing two flights of stairs. No subject was taking medications. Pulmonary function tests were performed according to ATS recommendations. Bronchoalveolar lavage was performed with a flexible fiberoptic brochoscope as described using a total of 300 ml of normal saline in 5,20 ml aliquots for each of three sites lavaged (Hunninghake et al., 1979). A total cell count was done in a hematocytometer, and cell differentials performed on cytocentrifuge slides stained with Diff-Quick (American Scientific, Edison, N J) and 500 cells counted. Viability was determined by trypan blue exclusion, and in all cases, recovered cells were >85% viable. Measurement o f oxidants. The spontaneous release of oxidants by the recovered alveolar macrophages was evaluated by incubating the macrophages in Hank's Balanced Salt Solution without phenol red at a concentration of 106/ml in Falcon 24-well plates (Becton-Dickinson, Mountain View, CA). Superoxide an-
DHEA [N ASBESTOSIS
147
ion release into the supernatant was measured by determining the reduction of 80 ~M ferricytochrome c (Sigma, St. Louis, MO) by 10 6 cells per hour at 550 nm using a spectrophotometer (Pick and Mizel, 1981). Units were expressed as the amount of cytochrome c reduced, calculated using a reduced-oxidized extinction coefficient of 21.1 mM- 1 c m - l by 106 cells per hour (Massey, 1959). Hydrogen peroxide release was measured by the change in absorbance at 610 nm by 10 6 cells per hour using the phenol red-horseradish peroxidase method (Pick and Keisari, 1980). Superoxide dismutase (0.5 mg/ml, Sigma) was added to reduce superoxide anion release, and catalase (4300 units/ml, Sigma) was added to reduce hydrogen peroxide release. DHEA (Sigma) was dissolved in ethanol and the various concentrations added in a volume of 3 pJ with an ethanol control. Ethanol vehicle as a control and DHEA in vehicle were added at time 0 and the incubation period was 1 hr. All experiments were performed in triplicate. Measurement of DHEA. The lavage fluid recovered from eight additional individuals (one normal, two asbestosis, five other lung disease including four interstitial lung disease) was examined by radioimmunoassay (RIA) for endogenous concentrations of DHEA, using the DHEA test set (No. DS-2100, Wien Laboratories, Succasunna, N J). Epithelial lining fluid (ELF) is diluted approximately 100 times by the technique of BAL (Rennard et al., 1986); therefore the protocol for the RIA was modified by extracting DHEA from a 20-ml aliquot of BAL fluid, which is 100 times the volume of serum the manufacturer recommends for extraction. The modification did not reduce the efficiency of extraction of DHEA, as recovery of a known amount of DHEA using the modified protocol was >99%. Statistics. All data are expressed as the mean _+ standard error of the mean. Comparisons between asbestosis with and without vehicle plus DHEA were made with the paired Student t test. The level of significance accepted was 0.05. RESULTS The nonsmoking asbestos workers with asbestosis had restrictive impairment on pulmonary function tests (Table I), pleural and parenchymal opacities on their chest radiographs, dyspnea upon climbing two flights of stairs, and 10/11 had rales on auscultation of their chest. The asbestos workers had increased cells recovered by bronchoalveolar lavage (asbestosis 380 - 56 x 103 cells/ml, normal
