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Increased Lower Respiratory Tract Iron Concentrations in Alkaloidal (“Crack”) Cocaine Users
Increased Lower Respiratory Tract Iron Concentrations in Alkaloidal (“Crack”) Cocaine Users* Tariq M. Janjua, MD; Amy E. Bohan, MD; and Lewis J. Wesselius, MD, FCCP
Study objective: We hypothesized that the use of inhaled alkaloidal (“crack”) cocaine could increase lung content of iron, either by inducing alveolar hemorrhage or by other mechanisms. Intrapulmonary accumulation of iron could promote chronic lung diseases in crack users. The goal of this study was to determine whether iron and ferritin content of alveolar macrophages or fluid recovered by BAL was increased in subjects using crack, compared with nonsmokers. Methods: BAL was performed in 31 volunteer subjects, including healthy nonsmokers (n ⴝ 7), subjects smoking crack alone (n ⴝ 7), as well as subjects smoking both crack and cigarettes (n ⴝ 7) or cigarettes alone (n ⴝ 10). Iron content of alveolar macrophages and BAL fluid was determined by a colorimetric method and ferritin content of alveolar macrophages, and BAL fluid was measured by a two-sided immunoradiometric method. Results: Alveolar macrophages recovered from crack users contained more iron than did alveolar macrophages from nonsmokers (25.4 ⴞ 2.9 nmol/106 vs 5.5 ⴞ 0.6 nmol/106 [mean ⴞ SE]; p < 0.01). There were similar increases in alveolar macrophage ferritin as well as BAL fluid iron and ferritin in crack users, compared with nonsmokers. BAL fluid ferritin concentrations in subjects smoking both crack and cigarettes were increased, compared with subjects smoking crack alone or cigarettes alone (p < 0.05). Conclusions: Use of crack increases intrapulmonary concentrations of iron and ferritin. Effects of crack on extracellular ferritin concentrations may be additive with effects of cigarette smoking. Although the mechanism(s) causing pulmonary iron accumulation were not identified by this study, it may be a result of occult alveolar hemorrhage or increased vascular permeability. The increase in lung iron burden in habitual crack users could promote chronic lung diseases in these subjects. (CHEST 2001; 119:422– 427) Key words: alveolar macrophage; cocaine; ferritin; iron
inhalation of alkaloidal (“crack”) cocaine can T heinduce a variety of acute pulmonary disorders, including alveolar hemorrhage, acute pulmonary edema, and interstitial pneumonitis.1 There is evidence that use of crack is also associated with the development of chronic lung injury, as indicated by decreased lung diffusing capacity.2– 4 However, little is known about the mechanism of lung injury associated with crack use. A prior study5 demonstrated occult alveolar hemorrhage at autopsy in 58% of crack users, even though death was due to unrelated causes. This observation suggests that occult alveolar hemorrhage *From the Pulmonary Section, Department of Medicine, Carl T. Hayden VA Medical Center, Phoenix, AZ. Supported by the VA Research Service. Manuscript received May 15, 2000; revision accepted September 14, 2000. Correspondence to: Lewis J. Wesselius, MD, Chief, Pulmonary Section, Carl T. Hayden VA Medical Center, 650 E. Indian School Rd, Phoenix, AZ 85012; e-mail: [email protected] 422
occurs frequently in subjects using crack. Repeated alveolar hemorrhage associated with crack use could increase lung iron burden as a result of accumulation of hemoglobin-derived iron. Alveolar hemorrhage and iron accumulation could contribute to the development of altered lung function in crack users. Chronic alveolar hemorrhage associated with idiopathic pulmonary hemosiderosis, for example, is accompanied by damage to the alveolar-capillary membrane and decreased lung diffusing capacity.6,7 Although the mechanisms contributing to the lung injury are uncertain, iron-catalyzed oxidative injury to alveolar structures may be a contributing factor. The clinical diagnosis of occult alveolar hemorrhage in patients has generally been based on the bronchoscopic recovery of alveolar macrophages that stain positively for hemosiderin-bound iron.8 However, prior studies9 –11 indicate that this finding may be transient and is not specific for alveolar hemorrhage. The staining technique used in the assessment Clinical Investigations
of occult alveolar hemorrhage is actually based on a semiquantitative assessment of alveolar macrophage iron content. A prior study11 compared this method of assessing cell iron content with direct measurement of alveolar macrophage iron and demonstrated a moderate correlation. However, alveolar macrophage iron content can be increased by factors other than alveolar hemorrhage, including cigarette smoking and mineral dust exposures, so that the finding of increased alveolar macrophage iron content is not specific for alveolar hemorrhage.11–14 Lung iron content is increased by various experimental lung injuries, including intrapulmonary instillation of silica and inhalation of ozone.15,16 These experimental lung injuries increase lung iron, at least in part, by increasing lung vascular permeability, resulting in an influx of serum-derived iron. Prior studies17,18 suggest that the use of crack also increases lung permeability, although this has not been demonstrated in all studies. Therefore, cocaineinduced alveolar hemorrhage, effects of cocaine on lung permeability, or possibly other effects of cocaine could lead to increased lung iron content. In order to assess whether crack use increases lung iron content, we specifically recruited a group of subjects using crack who denied use of tobacco or regular use of other illicit drugs. We also compared a group of subjects that smoked both cigarettes and crack with a group smoking comparable amounts of tobacco alone in order to assess whether these combined exposures might enhance lung iron accumulation. A control group of healthy nonsmokers was also included in the study. In this study, we report that crack users have a marked increase in lower respiratory tract content of iron and ferritin.
cigarette consumption are provided in Table 1. All subjects gave informed consent to participate in this study, and the study protocol was approved by the institutional human subjects review committee.
Materials and Methods
Results
Study Subjects Volunteer subjects were recruited by local advertisement and enrollment of subjects recently admitted to the substance abuse treatment unit. To be included in this study, subjects were required to have a history of recent smoking of crack (within 1 week) and prior regular use of inhaled cocaine for at least 6 months. Subjects were excluded from participation in this study if there was a reported history of regular IV drug use within the last year or if there was use of marijuana or other illicit drugs on a regular basis (more than once a week). Subjects using crack and not smoking cigarettes denied use of any tobacco products. Subjects smoking crack and tobacco or tobacco alone had smoked at least one pack of cigarettes daily for at least 7 years. Subjects recruited for the study were excluded if they admitted to regular use (more than once a week) of illicit drugs other than crack. The use of marijuana, if it occurred less than once a week, did not exclude volunteers. Some of the data on subjects smoking tobacco alone had been included in a prior study.19 The characteristics of all study subjects and quantification of crack and
BAL BAL was performed using methods that have been described previously.13 Briefly, the subjects received topical anesthesia to the oropharynx with tetracaine (2%) and were premedicated with midazolam. Bronchoscopy was performed transorally, and the bronchoscope was wedged initially into the right middle lobe. A total of 200 mL of saline solution was instilled in 50-mL aliquots, followed by immediate suctioning of each aliquot. The lavage procedure was subsequently repeated in the lingula, and the recovered fluid was pooled for analysis. Cells were recovered by centrifugation, and a total cell count was determined from an aliquot using a hemacytometer. A cell differential count was determined by counting 200 cells on a stained (Diff-Quick; American Scientific Products; McGaw Park, IL) cytotcentrifuge preparation. Iron and Ferritin Measurements The iron content of alveolar macrophages and BAL fluid was determined by a method based on the use of ferrozine, as described by Fish.20 This method involves the use of an ironreleasing reagent (0.6 N HCl and 2.25% weight/volume KMnO4), which releases iron complexed in biological samples. The sensitivity of this method is 1 g/dL. The ferritin content of alveolar macrophages and BAL fluid was measured using a solid-phase, two-sided immunoradiometric assay using antibodies to L-type ferritin (Hybritech; San Diego CA), as described by the manufacturer. This assay has a sensitivity of 0.7 ng/mL. Statistical Analysis Data are expressed as mean ⫾ SE. Differences between groups were analyzed by analysis of variance, and a correction for multiple comparisons was utilized (Student-Newman-Keuls method). In all tests, statistical significance was identified at the p ⬍ 0.05 level.
BAL Cell Recovery The number of cells recovered by BAL in study subjects is provided in Table 2. Total cell recovery by BAL in subjects smoking crack was increased compared with nonsmokers. There was a significantly
Table 1—Characteristics of Study Subjects* Variables
Age, yr
Cigarette Use, pack-yrs
Cocaine Months†
Control subjects Crack cocaine alone Crack cocaine and tobacco Tobacco
28 ⫾ 3 32 ⫾ 4 39 ⫾ 4 41 ⫾ 2
0 0 22 ⫾ 4 25 ⫾ 3
0 10 ⫾ 3 12 ⫾ 2 0
*Data are presented as mean ⫾ SD. †No. of months that crack cocaine was smoked on a regular basis. CHEST / 119 / 2 / FEBRUARY, 2001
423
Table 2—Cell Recovery by BAL* Variables
Total Cells (⫻ 106/L)
AM, %
Lymph, %
Neut, %
Eos, %
Nonsmokers Crack cocaine alone Crack cocaine and tobacco Tobacco
28.1 ⫾ 3.5 51.7 ⫾ 6.6† 134.3 ⫾ 29.0‡ 97.6 ⫾ 10.5‡
94.1 ⫾ 0.5 95.1 ⫾ 0.4 94.3 ⫾ 0.4 95.5 ⫾ 0.4
4.5 ⫾ 0.3 2.1 ⫾ 0.6 2.4 ⫾ 0.4 2.3 ⫾ 0.3
0.5 ⫾ 0.1 1.9 ⫾ 0.5 2.3 ⫾ 0.4 2.2 ⫾ 0.4
0.1 ⫾ 0.1 0.8 ⫾ 0.3 0.9 ⫾ 0.3 0.1 ⫾ 0.1
*AM ⫽ alveolar macrophage; Lymph ⫽ lymphocytes; Neut ⫽ neutrophils; Eos ⫽ eosinophils. †p ⬍ 0.05 compared with nonsmokers. ‡p ⬍ 0.01 compared with nonsmokers.
greater increase in total cell recovery in subjects smoking both cigarettes and crack, compared with subjects using only crack, although values were not significantly different from subjects smoking cigarettes alone (Table 2). In all smoking groups, the increase in total cell recovery was attributable predominantly to an increase in recovery of alveolar macrophages. There were no subjects with evidence of acute alveolar hemorrhage, as indicated by the finding of grossly bloody fluid or significant numbers of erythrocytes in recovered cell populations.
significant (Fig 1). Similarly, the ferritin content of alveolar macrophages recovered from crack users was also increased compared with alveolar macrophages from control subjects (Fig 2). There was approximately a fivefold increase in ferritin content of alveolar macrophages in crack users compared with control subjects. Again, the ferritin content of alveolar macrophages recovered from subjects using both crack and tobacco was higher when compared to subjects smoking crack alone or tobacco alone, although differences were not significant.
Alveolar Macrophage Iron and Ferritin
BAL Fluid Iron and Ferritin
The iron content of alveolar macrophages recovered from subjects using crack was significantly increased compared with control subjects (Fig 1). The mean iron content of alveolar macrophages in crack users was approximately fivefold greater than the iron content of alveolar macrophages recovered from nonsmokers. The alveolar macrophage iron content was higher in subjects using both crack and tobacco when compared to those smoking only crack or only cigarettes; however, the differences were not
The iron content of BAL fluid recovered from nonsmokers was ⬍ 10 ng/mL in all subjects, as noted in a prior study.19 The iron content of BAL fluid recovered from crack users was significantly increased compared with that present in control subjects (Fig 3). The iron concentrations in BAL fluid recovered from subjects using both crack and tobacco were higher than concentrations in subjects
Figure 1. Iron content of alveolar macrophages (AM) recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.01 compared with control subjects.
Figure 2. Ferritin content of alveolar macrophages recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.01 compared with control subjects. See Figure 1 for abbreviation.
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Clinical Investigations
subjects using both crack and tobacco was ⬎ 30-fold greater than concentrations in control subjects.
Discussion
Figure 3. Iron content of BAL fluid recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.01 compared with control subjects.
using crack alone or tobacco alone; however, differences were not statistically significant There were increases in BAL fluid concentrations of ferritin in subjects smoking crack compared with control subjects (Fig 4). The increases in extracellular ferritin in subjects smoking crack cocaine was approximately 17-fold compared with control subjects. There were even greater increases in ferritin concentrations in BAL fluid recovered from subjects using both crack and tobacco, which were significantly greater than concentrations in subjects smoking crack alone or tobacco alone (Fig 4). The mean concentration of ferritin in BAL fluid recovered from
Figure 4. Ferritin content of BAL fluid recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.001 compared with control subjects; ⫹ ⫽ p ⬍ 0.05 compared with subjects smoking only crack cocaine or tobacco alone.
The principal finding of this study is that crack users have an increased lower respiratory tract content of iron and ferritin, both within alveolar macrophages and in alveolar epithelial lining fluid. These findings extend prior reports11–14 that lung iron content is increased by cigarette smoking, mineral dust exposures, and in alveolar hemorrhage syndromes. The similar increase in lung content of both iron and ferritin suggests that most of the increased iron was ferritin bound. Although binding of iron to ferritin within cells provides partial protection against iron-catalyzed injury, there is experimental evidence20 that extracellular ferritin-bound iron can promote lung injury. Therefore, our findings suggest a mechanism whereby crack use could promote chronic lung injury. The increase in pulmonary iron concentrations in crack users that we noted could be caused by occult alveolar hemorrhage in these subjects. A prior study5 suggests that occult alveolar hemorrhage occurs commonly in crack users. We did not find evidence of acute alveolar hemorrhage, as indicated by the recovery of bloody BAL fluid in any of our subjects, although this does not rule out recent alveolar hemorrhage.10 The accumulation of iron within alveolar macrophages is consistent with alveolar hemorrhage but is not specific for this diagnosis. Another mechanisms that could increase alveolar macrophage content of iron would be increased lung epithelial permeability, which has been demonstrated in some studies17,18 of crack cocaine users. Alternatively, particulates accumulating in alveolar macrophages of crack cocaine users, which has also been previously noted,21–23 could increase cell iron content. Alveolar macrophages recovered from crack users demonstrate altered function, including decreased bacterial killing and impaired capacity to prevent tumor cell growth.24 Accumulation of iron in alveolar macrophages of crack users may contribute to these reported alterations in alveolar macrophage function. Iron accumulation has been shown to alter alveolar macrophage function, including cytokine expression, and can lead to cell injury.25,26 However, it is uncertain whether iron accumulation is the only cause of altered function in alveolar macrophages recovered from crack users. BAL fluid recovered from crack users contained significant amounts of extracellular ferritin, indicating increased iron and ferritin concentrations present in alveolar epithelial lining fluid. The finding CHEST / 119 / 2 / FEBRUARY, 2001
425
of increased extracellular ferritin is significant, since a prior study27 suggests that extracellular ferritin promotes lung injury in experimental animals. The effects of crack on extracellular ferritin accumulation within the lungs appeared to be additive to the effects of cigarette smoking, since subjects that smoked cigarettes and crack had significantly higher ferritin concentrations, compared with subjects smoking crack alone or a group of subjects smoking comparable amounts of tobacco alone. Since cigarette smoke mobilizes iron from extracellular ferritin, which can then potentially promote iron-catalyzed lung injury, there may be synergistic detrimental effects of exposure to both crack and cigarettes.28 Several possible factors may limit the conclusions of the current study, including the effect of the use other illicit drugs by study subjects, and the potential of inaccurate smoking information provided by study subjects. We screened all subjects and excluded subjects who reported regular use of other illicit drugs, although we did not exclude crack users with infrequent use of marijuana (less than once a week). In both groups of crack users, there were subjects who reported occasional use of marijuana. Therefore, it is possible that some iron accumulation in the lungs of subjects smoking crack was attributable to the concurrent use of marijuana. It is also possible that some subjects may have used other illicit inhaled or IV agents that were not reported and which could influence our results. A prior study29 suggests that IV drugs may also lead to an increase in iron content of lung macrophages. It is also possible that smoking histories obtained from study subjects were not accurate, making it difficult to make conclusions about the relative effects of crack and cigarette smoking on lung iron accumulation. Prior studies30 have implicated increased pulmonary concentrations of iron in the development of lung neoplasms, presumably by enhancing generation of hydroxyl radicals that can promote DNA injury. Systemic iron overload associated with thalassemia has also been shown to promote development of a restrictive lung disease and hypoxemia consistent with pulmonary fibrosis.31 Although these prior studies support the concept that iron accumulation within the lungs can promote lung disease, the exact role of intrapulmonary iron in the pathogenesis of lung disease in crack users is uncertain. In summary, the current study demonstrates that lung iron content is markedly increased in habitual crack users. Although the mechanism(s) contributing to the accumulation of iron are not clear from these studies, subclinical alveolar hemorrhage or increased lung permeability could be contributing factors. The increase in pulmonary iron burden in crack users 426
may contribute to impaired alveolar macrophage function in these subjects and could potentially promote the development of chronic pulmonary diseases. References 1 Haim DY, Lippmann ML, Goldberg SK, et al. The pulmonary complications of crack cocaine: a comprehensive review. Chest 1995; 107:233–240 2 Weiss RD, Goldenheim PD, Mirin SM, et al. Pulmonary dysfunction in cocaine smokers. Am J Psychiatry 1981; 138: 1110 –1112 3 Ithonen J, Scholl S, Glassroth J. Pulmonary dysfunction in free-base cocaine users. Arch Intern Med 1984; 144:2195– 2197 4 Tashkin DP, Khalsa ME, Gorelick D, et al. Pulmonary status of habitual cocaine smokers. Am Rev Respir Dis 1992; 145:92–100 5 Bailey ME, Fraire AE, Greenberg SD, et al. Pulmonary histopathology in cocaine abusers. Hum Pathol 1994; 25:203–207 6 Roberts LN, Montessori G, Patterson JG. Idiopathic pulmonary hemosiderosis: case report with pulmonary function tests and lveolar macrophage function and cytokine production. Am Rev Respir Dis 1972; 106:904 –908 7 Corrin B, Jagusch M, Dewar A, et al. Fine structural changes in idiopathic pulmonary hemosiderosis. J Pathol 1987; 153: 249 –256 8 De Lassence A, Fleury-Feith J, Escudier E, et al. Alveolar hemorrhage: diagnostic criteria and results in 194 immunocompromised hosts. Am J Respir Crit Care Med 1995; 151:157–163 9 Grebski E, Hess T, Hold G, et al. Diagnostic value of hemosiderin-containing macrophages in bronchoalveolar lavage. Chest 1992; 102:1794 –1799 10 Sherman JM, Winnie G, Thomassen MJ, et al. Time course of hemosiderin production and clearance by human pulmonary macrophages. Chest 1984; 86:409 – 411 11 Corhay JL, Wever G, Bury T, et al. Iron content in human alveolar macrophages. Eur Respir J 1992; 5:804 – 809 12 Mateos F, Brock JH, Perez-Arellano JL. Iron metabolism in the lower respiratory tract. Thorax 1998; 53:594 – 600 13 Wesselius LJ, Flowers CH, Skikne BS. Alveolar macrophage content of isoferritins and transferrin: comparison of nonsmokers and smokers with and without chronic airflow obstruction. Am Rev Respir Dis 1992; 145:311–316 14 Guest L. The endogenous iron content, by Mossbauer spectroscopy, of human lungs: II. Lungs from various occupational groups. Ann Occup Hyg 1978; 21:151–157 15 Mills PC, Roberts CA, Smith NC. Effects of ozone and airway inflammation on glutathione status and iron homeostasis in the lungs of horses. Am J Veterinary Res 1996; 57:1359 –1363 16 Ghio AJ, Jaskot RH, Hatch GE. Lung injury after silica instillation is associated with an accumulation of iron in rats. Am J Physiol 1994; 267(6 pt 1)L686 –L692: 17 Susskind H, Weber DA, Volkow ND, et al. Increased lung permeability following long-term use of free-base cocaine (crack). Chest 1991; 100:903–909 18 Tashkin DP, Kleerup EC, Hoh CK, et al. Effects of “crack” cocaine on pulmonary alveolar permeability. Chest 1997; 112:327–335 19 Nelson ME, O’Brien-Ladner AR, Wesselius LJ. Regional variation in iron and iron-binding proteins within the lungs of smokers. Am J Respir Crit Care Med 1995; 153:1353–1358 20 Fish WW. Rapid colorimetric micromethod for the quantitaClinical Investigations
21 22
23 24 25
tion of complexed iron in biologic samples. Methods Enzymol 1988; 158:357–364 Greenbaum E, Copeland A, Grewal R. Blackened bronchoalveolar lavage fluid in crack smokers. Am J Clin Pathol 1993; 100:481– 487 Wesselius LJ, Smirnov IM, Nelson ME, et al. Alveolar macrophages accumulate iron and ferritin after in vivo exposure to iron or tungsten dusts. J Lab Clin Med 1996; 127:401– 409 Giovagnoli MR, Alderisio M, Cenci M, et al. Carbon and hemosiderin-laden macrophages in sputum of traffic policeman exposed to air pollution. Arch Environ Health 1999; 54:284 –290 Baldwin GC, Tashkin DP, Buckley DM, et al. Marijuana and cocaine impair alveolar macrophage function and cytokine production. Am J Respir Crit Care Med 1997; 156:1606 –1613 O’Brien-Ladner AR, Blumer BM, Wesselius LJ. Differential regulation of human alveolar macrophage-derived interleukin-1 and tumor necrosis factor-␣ by iron. J Lab Clin Med 1998; 132:497–506
26 Fuhrman B, Oiknine J, Aviram M. Iron induces lipid peroxidation in cultured macrophages, increases their ability to oxidatively modify LDL, and affects their secretory properties. Atherosclerosis 1994; 111:65–78 27 Folkesson HG, Leanderson P, Westrom BR, et al. Increased lung to blood passage of polyethylene glycols after intratracheal instillation of ferritin and asbestos fibers in the rat. Eur Respir J 1993; 6:96 –101 28 Lapenna D, De Gioia S, Mezzetti A, et al. Cigarette smoke, ferritin and lipid peroxidation. Am J Respir Crit Care Med 1995; 151:431– 435 29 Lockemann U, Pueschel K. Siderophages in the lung of drug addicts. Forensic Sci Int 1993; 59:169 –175 30 Weinberg ED. Association of iron with respiratory tract neoplasia. J Trace Elem Exp Med 1993; 6:117–123 31 Cooper DM, Mansell AL, Weiner MA, et al. Low lung capacity and hypoxemia in children with thalassemia major. Am Rev Respir Dis 1980; 121:639 – 646
CHEST / 119 / 2 / FEBRUARY, 2001
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Study objective: We hypothesized that the use of inhaled alkaloidal (“crack”) cocaine could increase lung content of iron, either by inducing alveolar hemorrhage or by other mechanisms. Intrapulmonary accumulation of iron could promote chronic lung diseases in crack users. The goal of this study was to determine whether iron and ferritin content of alveolar macrophages or fluid recovered by BAL was increased in subjects using crack, compared with nonsmokers. Methods: BAL was performed in 31 volunteer subjects, including healthy nonsmokers (n ⴝ 7), subjects smoking crack alone (n ⴝ 7), as well as subjects smoking both crack and cigarettes (n ⴝ 7) or cigarettes alone (n ⴝ 10). Iron content of alveolar macrophages and BAL fluid was determined by a colorimetric method and ferritin content of alveolar macrophages, and BAL fluid was measured by a two-sided immunoradiometric method. Results: Alveolar macrophages recovered from crack users contained more iron than did alveolar macrophages from nonsmokers (25.4 ⴞ 2.9 nmol/106 vs 5.5 ⴞ 0.6 nmol/106 [mean ⴞ SE]; p < 0.01). There were similar increases in alveolar macrophage ferritin as well as BAL fluid iron and ferritin in crack users, compared with nonsmokers. BAL fluid ferritin concentrations in subjects smoking both crack and cigarettes were increased, compared with subjects smoking crack alone or cigarettes alone (p < 0.05). Conclusions: Use of crack increases intrapulmonary concentrations of iron and ferritin. Effects of crack on extracellular ferritin concentrations may be additive with effects of cigarette smoking. Although the mechanism(s) causing pulmonary iron accumulation were not identified by this study, it may be a result of occult alveolar hemorrhage or increased vascular permeability. The increase in lung iron burden in habitual crack users could promote chronic lung diseases in these subjects. (CHEST 2001; 119:422– 427) Key words: alveolar macrophage; cocaine; ferritin; iron
inhalation of alkaloidal (“crack”) cocaine can T heinduce a variety of acute pulmonary disorders, including alveolar hemorrhage, acute pulmonary edema, and interstitial pneumonitis.1 There is evidence that use of crack is also associated with the development of chronic lung injury, as indicated by decreased lung diffusing capacity.2– 4 However, little is known about the mechanism of lung injury associated with crack use. A prior study5 demonstrated occult alveolar hemorrhage at autopsy in 58% of crack users, even though death was due to unrelated causes. This observation suggests that occult alveolar hemorrhage *From the Pulmonary Section, Department of Medicine, Carl T. Hayden VA Medical Center, Phoenix, AZ. Supported by the VA Research Service. Manuscript received May 15, 2000; revision accepted September 14, 2000. Correspondence to: Lewis J. Wesselius, MD, Chief, Pulmonary Section, Carl T. Hayden VA Medical Center, 650 E. Indian School Rd, Phoenix, AZ 85012; e-mail: [email protected] 422
occurs frequently in subjects using crack. Repeated alveolar hemorrhage associated with crack use could increase lung iron burden as a result of accumulation of hemoglobin-derived iron. Alveolar hemorrhage and iron accumulation could contribute to the development of altered lung function in crack users. Chronic alveolar hemorrhage associated with idiopathic pulmonary hemosiderosis, for example, is accompanied by damage to the alveolar-capillary membrane and decreased lung diffusing capacity.6,7 Although the mechanisms contributing to the lung injury are uncertain, iron-catalyzed oxidative injury to alveolar structures may be a contributing factor. The clinical diagnosis of occult alveolar hemorrhage in patients has generally been based on the bronchoscopic recovery of alveolar macrophages that stain positively for hemosiderin-bound iron.8 However, prior studies9 –11 indicate that this finding may be transient and is not specific for alveolar hemorrhage. The staining technique used in the assessment Clinical Investigations
of occult alveolar hemorrhage is actually based on a semiquantitative assessment of alveolar macrophage iron content. A prior study11 compared this method of assessing cell iron content with direct measurement of alveolar macrophage iron and demonstrated a moderate correlation. However, alveolar macrophage iron content can be increased by factors other than alveolar hemorrhage, including cigarette smoking and mineral dust exposures, so that the finding of increased alveolar macrophage iron content is not specific for alveolar hemorrhage.11–14 Lung iron content is increased by various experimental lung injuries, including intrapulmonary instillation of silica and inhalation of ozone.15,16 These experimental lung injuries increase lung iron, at least in part, by increasing lung vascular permeability, resulting in an influx of serum-derived iron. Prior studies17,18 suggest that the use of crack also increases lung permeability, although this has not been demonstrated in all studies. Therefore, cocaineinduced alveolar hemorrhage, effects of cocaine on lung permeability, or possibly other effects of cocaine could lead to increased lung iron content. In order to assess whether crack use increases lung iron content, we specifically recruited a group of subjects using crack who denied use of tobacco or regular use of other illicit drugs. We also compared a group of subjects that smoked both cigarettes and crack with a group smoking comparable amounts of tobacco alone in order to assess whether these combined exposures might enhance lung iron accumulation. A control group of healthy nonsmokers was also included in the study. In this study, we report that crack users have a marked increase in lower respiratory tract content of iron and ferritin.
cigarette consumption are provided in Table 1. All subjects gave informed consent to participate in this study, and the study protocol was approved by the institutional human subjects review committee.
Materials and Methods
Results
Study Subjects Volunteer subjects were recruited by local advertisement and enrollment of subjects recently admitted to the substance abuse treatment unit. To be included in this study, subjects were required to have a history of recent smoking of crack (within 1 week) and prior regular use of inhaled cocaine for at least 6 months. Subjects were excluded from participation in this study if there was a reported history of regular IV drug use within the last year or if there was use of marijuana or other illicit drugs on a regular basis (more than once a week). Subjects using crack and not smoking cigarettes denied use of any tobacco products. Subjects smoking crack and tobacco or tobacco alone had smoked at least one pack of cigarettes daily for at least 7 years. Subjects recruited for the study were excluded if they admitted to regular use (more than once a week) of illicit drugs other than crack. The use of marijuana, if it occurred less than once a week, did not exclude volunteers. Some of the data on subjects smoking tobacco alone had been included in a prior study.19 The characteristics of all study subjects and quantification of crack and
BAL BAL was performed using methods that have been described previously.13 Briefly, the subjects received topical anesthesia to the oropharynx with tetracaine (2%) and were premedicated with midazolam. Bronchoscopy was performed transorally, and the bronchoscope was wedged initially into the right middle lobe. A total of 200 mL of saline solution was instilled in 50-mL aliquots, followed by immediate suctioning of each aliquot. The lavage procedure was subsequently repeated in the lingula, and the recovered fluid was pooled for analysis. Cells were recovered by centrifugation, and a total cell count was determined from an aliquot using a hemacytometer. A cell differential count was determined by counting 200 cells on a stained (Diff-Quick; American Scientific Products; McGaw Park, IL) cytotcentrifuge preparation. Iron and Ferritin Measurements The iron content of alveolar macrophages and BAL fluid was determined by a method based on the use of ferrozine, as described by Fish.20 This method involves the use of an ironreleasing reagent (0.6 N HCl and 2.25% weight/volume KMnO4), which releases iron complexed in biological samples. The sensitivity of this method is 1 g/dL. The ferritin content of alveolar macrophages and BAL fluid was measured using a solid-phase, two-sided immunoradiometric assay using antibodies to L-type ferritin (Hybritech; San Diego CA), as described by the manufacturer. This assay has a sensitivity of 0.7 ng/mL. Statistical Analysis Data are expressed as mean ⫾ SE. Differences between groups were analyzed by analysis of variance, and a correction for multiple comparisons was utilized (Student-Newman-Keuls method). In all tests, statistical significance was identified at the p ⬍ 0.05 level.
BAL Cell Recovery The number of cells recovered by BAL in study subjects is provided in Table 2. Total cell recovery by BAL in subjects smoking crack was increased compared with nonsmokers. There was a significantly
Table 1—Characteristics of Study Subjects* Variables
Age, yr
Cigarette Use, pack-yrs
Cocaine Months†
Control subjects Crack cocaine alone Crack cocaine and tobacco Tobacco
28 ⫾ 3 32 ⫾ 4 39 ⫾ 4 41 ⫾ 2
0 0 22 ⫾ 4 25 ⫾ 3
0 10 ⫾ 3 12 ⫾ 2 0
*Data are presented as mean ⫾ SD. †No. of months that crack cocaine was smoked on a regular basis. CHEST / 119 / 2 / FEBRUARY, 2001
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Table 2—Cell Recovery by BAL* Variables
Total Cells (⫻ 106/L)
AM, %
Lymph, %
Neut, %
Eos, %
Nonsmokers Crack cocaine alone Crack cocaine and tobacco Tobacco
28.1 ⫾ 3.5 51.7 ⫾ 6.6† 134.3 ⫾ 29.0‡ 97.6 ⫾ 10.5‡
94.1 ⫾ 0.5 95.1 ⫾ 0.4 94.3 ⫾ 0.4 95.5 ⫾ 0.4
4.5 ⫾ 0.3 2.1 ⫾ 0.6 2.4 ⫾ 0.4 2.3 ⫾ 0.3
0.5 ⫾ 0.1 1.9 ⫾ 0.5 2.3 ⫾ 0.4 2.2 ⫾ 0.4
0.1 ⫾ 0.1 0.8 ⫾ 0.3 0.9 ⫾ 0.3 0.1 ⫾ 0.1
*AM ⫽ alveolar macrophage; Lymph ⫽ lymphocytes; Neut ⫽ neutrophils; Eos ⫽ eosinophils. †p ⬍ 0.05 compared with nonsmokers. ‡p ⬍ 0.01 compared with nonsmokers.
greater increase in total cell recovery in subjects smoking both cigarettes and crack, compared with subjects using only crack, although values were not significantly different from subjects smoking cigarettes alone (Table 2). In all smoking groups, the increase in total cell recovery was attributable predominantly to an increase in recovery of alveolar macrophages. There were no subjects with evidence of acute alveolar hemorrhage, as indicated by the finding of grossly bloody fluid or significant numbers of erythrocytes in recovered cell populations.
significant (Fig 1). Similarly, the ferritin content of alveolar macrophages recovered from crack users was also increased compared with alveolar macrophages from control subjects (Fig 2). There was approximately a fivefold increase in ferritin content of alveolar macrophages in crack users compared with control subjects. Again, the ferritin content of alveolar macrophages recovered from subjects using both crack and tobacco was higher when compared to subjects smoking crack alone or tobacco alone, although differences were not significant.
Alveolar Macrophage Iron and Ferritin
BAL Fluid Iron and Ferritin
The iron content of alveolar macrophages recovered from subjects using crack was significantly increased compared with control subjects (Fig 1). The mean iron content of alveolar macrophages in crack users was approximately fivefold greater than the iron content of alveolar macrophages recovered from nonsmokers. The alveolar macrophage iron content was higher in subjects using both crack and tobacco when compared to those smoking only crack or only cigarettes; however, the differences were not
The iron content of BAL fluid recovered from nonsmokers was ⬍ 10 ng/mL in all subjects, as noted in a prior study.19 The iron content of BAL fluid recovered from crack users was significantly increased compared with that present in control subjects (Fig 3). The iron concentrations in BAL fluid recovered from subjects using both crack and tobacco were higher than concentrations in subjects
Figure 1. Iron content of alveolar macrophages (AM) recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.01 compared with control subjects.
Figure 2. Ferritin content of alveolar macrophages recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.01 compared with control subjects. See Figure 1 for abbreviation.
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subjects using both crack and tobacco was ⬎ 30-fold greater than concentrations in control subjects.
Discussion
Figure 3. Iron content of BAL fluid recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.01 compared with control subjects.
using crack alone or tobacco alone; however, differences were not statistically significant There were increases in BAL fluid concentrations of ferritin in subjects smoking crack compared with control subjects (Fig 4). The increases in extracellular ferritin in subjects smoking crack cocaine was approximately 17-fold compared with control subjects. There were even greater increases in ferritin concentrations in BAL fluid recovered from subjects using both crack and tobacco, which were significantly greater than concentrations in subjects smoking crack alone or tobacco alone (Fig 4). The mean concentration of ferritin in BAL fluid recovered from
Figure 4. Ferritin content of BAL fluid recovered from control (nonsmoking) subjects, subjects smoking only crack cocaine, subjects smoking crack cocaine and tobacco, and subjects smoking only tobacco. * ⫽ p ⬍ 0.001 compared with control subjects; ⫹ ⫽ p ⬍ 0.05 compared with subjects smoking only crack cocaine or tobacco alone.
The principal finding of this study is that crack users have an increased lower respiratory tract content of iron and ferritin, both within alveolar macrophages and in alveolar epithelial lining fluid. These findings extend prior reports11–14 that lung iron content is increased by cigarette smoking, mineral dust exposures, and in alveolar hemorrhage syndromes. The similar increase in lung content of both iron and ferritin suggests that most of the increased iron was ferritin bound. Although binding of iron to ferritin within cells provides partial protection against iron-catalyzed injury, there is experimental evidence20 that extracellular ferritin-bound iron can promote lung injury. Therefore, our findings suggest a mechanism whereby crack use could promote chronic lung injury. The increase in pulmonary iron concentrations in crack users that we noted could be caused by occult alveolar hemorrhage in these subjects. A prior study5 suggests that occult alveolar hemorrhage occurs commonly in crack users. We did not find evidence of acute alveolar hemorrhage, as indicated by the recovery of bloody BAL fluid in any of our subjects, although this does not rule out recent alveolar hemorrhage.10 The accumulation of iron within alveolar macrophages is consistent with alveolar hemorrhage but is not specific for this diagnosis. Another mechanisms that could increase alveolar macrophage content of iron would be increased lung epithelial permeability, which has been demonstrated in some studies17,18 of crack cocaine users. Alternatively, particulates accumulating in alveolar macrophages of crack cocaine users, which has also been previously noted,21–23 could increase cell iron content. Alveolar macrophages recovered from crack users demonstrate altered function, including decreased bacterial killing and impaired capacity to prevent tumor cell growth.24 Accumulation of iron in alveolar macrophages of crack users may contribute to these reported alterations in alveolar macrophage function. Iron accumulation has been shown to alter alveolar macrophage function, including cytokine expression, and can lead to cell injury.25,26 However, it is uncertain whether iron accumulation is the only cause of altered function in alveolar macrophages recovered from crack users. BAL fluid recovered from crack users contained significant amounts of extracellular ferritin, indicating increased iron and ferritin concentrations present in alveolar epithelial lining fluid. The finding CHEST / 119 / 2 / FEBRUARY, 2001
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of increased extracellular ferritin is significant, since a prior study27 suggests that extracellular ferritin promotes lung injury in experimental animals. The effects of crack on extracellular ferritin accumulation within the lungs appeared to be additive to the effects of cigarette smoking, since subjects that smoked cigarettes and crack had significantly higher ferritin concentrations, compared with subjects smoking crack alone or a group of subjects smoking comparable amounts of tobacco alone. Since cigarette smoke mobilizes iron from extracellular ferritin, which can then potentially promote iron-catalyzed lung injury, there may be synergistic detrimental effects of exposure to both crack and cigarettes.28 Several possible factors may limit the conclusions of the current study, including the effect of the use other illicit drugs by study subjects, and the potential of inaccurate smoking information provided by study subjects. We screened all subjects and excluded subjects who reported regular use of other illicit drugs, although we did not exclude crack users with infrequent use of marijuana (less than once a week). In both groups of crack users, there were subjects who reported occasional use of marijuana. Therefore, it is possible that some iron accumulation in the lungs of subjects smoking crack was attributable to the concurrent use of marijuana. It is also possible that some subjects may have used other illicit inhaled or IV agents that were not reported and which could influence our results. A prior study29 suggests that IV drugs may also lead to an increase in iron content of lung macrophages. It is also possible that smoking histories obtained from study subjects were not accurate, making it difficult to make conclusions about the relative effects of crack and cigarette smoking on lung iron accumulation. Prior studies30 have implicated increased pulmonary concentrations of iron in the development of lung neoplasms, presumably by enhancing generation of hydroxyl radicals that can promote DNA injury. Systemic iron overload associated with thalassemia has also been shown to promote development of a restrictive lung disease and hypoxemia consistent with pulmonary fibrosis.31 Although these prior studies support the concept that iron accumulation within the lungs can promote lung disease, the exact role of intrapulmonary iron in the pathogenesis of lung disease in crack users is uncertain. In summary, the current study demonstrates that lung iron content is markedly increased in habitual crack users. Although the mechanism(s) contributing to the accumulation of iron are not clear from these studies, subclinical alveolar hemorrhage or increased lung permeability could be contributing factors. The increase in pulmonary iron burden in crack users 426
may contribute to impaired alveolar macrophage function in these subjects and could potentially promote the development of chronic pulmonary diseases. References 1 Haim DY, Lippmann ML, Goldberg SK, et al. The pulmonary complications of crack cocaine: a comprehensive review. Chest 1995; 107:233–240 2 Weiss RD, Goldenheim PD, Mirin SM, et al. Pulmonary dysfunction in cocaine smokers. Am J Psychiatry 1981; 138: 1110 –1112 3 Ithonen J, Scholl S, Glassroth J. Pulmonary dysfunction in free-base cocaine users. Arch Intern Med 1984; 144:2195– 2197 4 Tashkin DP, Khalsa ME, Gorelick D, et al. Pulmonary status of habitual cocaine smokers. Am Rev Respir Dis 1992; 145:92–100 5 Bailey ME, Fraire AE, Greenberg SD, et al. Pulmonary histopathology in cocaine abusers. Hum Pathol 1994; 25:203–207 6 Roberts LN, Montessori G, Patterson JG. Idiopathic pulmonary hemosiderosis: case report with pulmonary function tests and lveolar macrophage function and cytokine production. Am Rev Respir Dis 1972; 106:904 –908 7 Corrin B, Jagusch M, Dewar A, et al. Fine structural changes in idiopathic pulmonary hemosiderosis. J Pathol 1987; 153: 249 –256 8 De Lassence A, Fleury-Feith J, Escudier E, et al. Alveolar hemorrhage: diagnostic criteria and results in 194 immunocompromised hosts. Am J Respir Crit Care Med 1995; 151:157–163 9 Grebski E, Hess T, Hold G, et al. Diagnostic value of hemosiderin-containing macrophages in bronchoalveolar lavage. Chest 1992; 102:1794 –1799 10 Sherman JM, Winnie G, Thomassen MJ, et al. Time course of hemosiderin production and clearance by human pulmonary macrophages. Chest 1984; 86:409 – 411 11 Corhay JL, Wever G, Bury T, et al. Iron content in human alveolar macrophages. Eur Respir J 1992; 5:804 – 809 12 Mateos F, Brock JH, Perez-Arellano JL. Iron metabolism in the lower respiratory tract. Thorax 1998; 53:594 – 600 13 Wesselius LJ, Flowers CH, Skikne BS. Alveolar macrophage content of isoferritins and transferrin: comparison of nonsmokers and smokers with and without chronic airflow obstruction. Am Rev Respir Dis 1992; 145:311–316 14 Guest L. The endogenous iron content, by Mossbauer spectroscopy, of human lungs: II. Lungs from various occupational groups. Ann Occup Hyg 1978; 21:151–157 15 Mills PC, Roberts CA, Smith NC. Effects of ozone and airway inflammation on glutathione status and iron homeostasis in the lungs of horses. Am J Veterinary Res 1996; 57:1359 –1363 16 Ghio AJ, Jaskot RH, Hatch GE. Lung injury after silica instillation is associated with an accumulation of iron in rats. Am J Physiol 1994; 267(6 pt 1)L686 –L692: 17 Susskind H, Weber DA, Volkow ND, et al. Increased lung permeability following long-term use of free-base cocaine (crack). Chest 1991; 100:903–909 18 Tashkin DP, Kleerup EC, Hoh CK, et al. Effects of “crack” cocaine on pulmonary alveolar permeability. Chest 1997; 112:327–335 19 Nelson ME, O’Brien-Ladner AR, Wesselius LJ. Regional variation in iron and iron-binding proteins within the lungs of smokers. Am J Respir Crit Care Med 1995; 153:1353–1358 20 Fish WW. Rapid colorimetric micromethod for the quantitaClinical Investigations
21 22
23 24 25
tion of complexed iron in biologic samples. Methods Enzymol 1988; 158:357–364 Greenbaum E, Copeland A, Grewal R. Blackened bronchoalveolar lavage fluid in crack smokers. Am J Clin Pathol 1993; 100:481– 487 Wesselius LJ, Smirnov IM, Nelson ME, et al. Alveolar macrophages accumulate iron and ferritin after in vivo exposure to iron or tungsten dusts. J Lab Clin Med 1996; 127:401– 409 Giovagnoli MR, Alderisio M, Cenci M, et al. Carbon and hemosiderin-laden macrophages in sputum of traffic policeman exposed to air pollution. Arch Environ Health 1999; 54:284 –290 Baldwin GC, Tashkin DP, Buckley DM, et al. Marijuana and cocaine impair alveolar macrophage function and cytokine production. Am J Respir Crit Care Med 1997; 156:1606 –1613 O’Brien-Ladner AR, Blumer BM, Wesselius LJ. Differential regulation of human alveolar macrophage-derived interleukin-1 and tumor necrosis factor-␣ by iron. J Lab Clin Med 1998; 132:497–506
26 Fuhrman B, Oiknine J, Aviram M. Iron induces lipid peroxidation in cultured macrophages, increases their ability to oxidatively modify LDL, and affects their secretory properties. Atherosclerosis 1994; 111:65–78 27 Folkesson HG, Leanderson P, Westrom BR, et al. Increased lung to blood passage of polyethylene glycols after intratracheal instillation of ferritin and asbestos fibers in the rat. Eur Respir J 1993; 6:96 –101 28 Lapenna D, De Gioia S, Mezzetti A, et al. Cigarette smoke, ferritin and lipid peroxidation. Am J Respir Crit Care Med 1995; 151:431– 435 29 Lockemann U, Pueschel K. Siderophages in the lung of drug addicts. Forensic Sci Int 1993; 59:169 –175 30 Weinberg ED. Association of iron with respiratory tract neoplasia. J Trace Elem Exp Med 1993; 6:117–123 31 Cooper DM, Mansell AL, Weiner MA, et al. Low lung capacity and hypoxemia in children with thalassemia major. Am Rev Respir Dis 1980; 121:639 – 646
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