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Alveolar lesions induced by systemic administration of cocaine to rats
Toxicology Letters 110 (1999) 113 – 118 www.elsevier.com/locate/toxlet
Alveolar lesions induced by systemic administration of cocaine to rats Rosario Barroso-Moguel a,*, Juana Villeda-Herna´ndez a, Marisela Me´ndez-Armenta a, Abel Santamarı´a b, Sonia Galva´n-Arzate c a
Laboratory of Cellular Neuromorphology, National Institute of Neurology and Neurosurgery, A6. Insurgentes Sur 3877, Mexico D.F. 14269, Mexico b Laboratory of Excitatory Aminoacids, National Institute of Neurology and Neurosurgery, A6. Insurgentes Sur 3877, Mexico D.F. 14269, Mexico c Department of Neurochemistry, National Institute of Neurology and Neurosurgery, A6. Insurgentes Sur 3877, Mexico D.F. 14269, Mexico Received 6 April 1999; accepted 28 July 1999
Abstract In this work, alveolar lesions induced after systemic administration of cocaine (30 mg/kg per day, i.p.) to rats were evaluated both by light microscope analysis for morphological assessment as well as by measurement of the alveolar area as a quantitative index of the alveolar damage. Rats were examined after different times of exposure: 7, 15, 30, 45, 60 and 75 days. The histopathological evaluation of cocaine-treated rats revealed a remarkable thickening in some interalveolar septa, with interstitial hemorrhages, progressive thrombosis and transformation of reticular and elastic fibers into diffuse fibrosis. A significant decrease of the alveolar area was also observed. These findings are indicative of severe changes in capillaries, alveoli and bronchiole after cocaine exposure, which in turn may progressively disrupt the general function of the lungs. Differential mechanisms of systemic toxicity after cocaine exposure are discussed. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cocaine; Alveoli; Lungs; Bronchiole; Macrophages; Edema; Fibrosis
1. Introduction Cocaine is a well-known addictive drug exhibiting a lot of adverse effects. Cocaine and related metabolites have been shown to be responsible of * Corresponding author. Tel.: +52-5-606-3822; fax: +525-528-0095. E-mail address: [email protected] (R. Barroso-Moguel)
several toxic actions occurring in the brain, liver, kidneys and testis, among others (Fleming et al., 1990; Barroso-Moguel et al., 1994, 1995, 1997). This drug has been demonstrated to interfere with DNA synthesis (Anderson-Brown et al., 1990) and may also induce seizures and lethality associated with excitatory aminoacid and dopamine receptors in the brain (Rockhold et al., 1991; Shimosato et al., 1995). It is also known that one
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 9 9 ) 0 0 1 4 8 - 4
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of the organs with major affinity for this drug is the lung. Moreover, cocaine is also capable to induce morphological and functional changes in human lungs, such as increased number of capillaries and disrupted permeability, leading to fibrosis, hemorrhage, congestion and intra-alveolar edema, also exhibiting an inflammatory infiltrate consisting of neutrophils, lymphocytes, macrophages and eosinophils (Bailey et al., 1994). However, only little information is available concerning the morphological alterations of alveoli produced after the systemic administration of cocaine to animals. In order to provide additional information on the toxic action of cocaine as a suitable model of cocaine intoxication in humans (Barroso-Moguel et al., 1997), this study was devoted to describe and compare the morphological alveolar alterations produced by this drug at different times after its systemic administration to rats as assessed by light microscopy. Alveolar areas were also analyzed as a quantitative index of cocaineinduced lung alterations.
2. Materials and methods
2.3. Cocaine treatment Control rats received saline i.p. daily (N= 36). Treated animals (N= 36) received an aqueous solution of cocaine hydrochloride (30 mg/kg per day, i.p.) for different periods of time (7, 15, 30, 45, 60 or 75 days). Similar doses have been previously used for the study of long-term effects of cocaine in rats (Church et al., 1988; Dow-Edwards, 1989; Barroso-Moguel et al., 1994, 1995, 1997). Total amounts of cocaine received by animals were 210, 450, 900, 1350, 1800 or 2250 mg/kg during the respective periods of administration.
2.4. Morphological examination Rats from both groups were anesthetized with 3.5% chloral hydrate i.p. and then perfused transcardially at a constant pressure on days 7, 15, 30, 45, 60 and 75 of treatment. Lungs were then dissected and fixed in 10% formalin during 15 days. Lung sections were paraffin-embedded and sectioned as 4–5 mm thin slices. Hematoxilineosin and Masson’s thrichrome stains were applied (Luna, 1960). Sections were examined with a Zeiss light photomicroscope (Fomi III).
2.1. Reagents Cocaine hydrochloride (C17H22CINO4) was obtained from the Department of Psychotropic Drugs at the Ministry of Health (Mexico). Formaldehyde and staining reagents were obtained from Mallinckrodt/Baker (Mexico). All other reagents were from E. Merck (Mexico).
2.2. Animals Male Wistar bred-in-house rats (225 – 230 g) were used throughout the study. Animals were housed in acrylic box cages and provided with standard rodent Chow (Purine Chow) and water ad libitum. Animals were maintained under conditions of constant temperature (259 3°C), humidity (50 910%) and lighting (12:12 h, light:dark cycle).
2.5. Measurement of al6eolar area Alveolar areas were analysed employing a digital analysis system (Zidas; Carl Zeiss) and a micrograph amplifier (1:10). Final magnification for measurement of areas was 400×. Nine fields per section (nine sections per rat), all randomly selected, were measured and integrated. Final areas were expressed in mm2 as mean 9 standard error (S.E.).
2.6. Statistical analysis Alveolar areas from control and cocaine-treated rats were statistically compared by Student’s ttest. Data were logaritmically transformed in order to equalize the variance prior t-testing.
R. Barroso-Moguel et al. / Toxicology Letters 110 (1999) 113–118
3. Results
3.1. Morphological findings Cocaine-treated rats exhibited dyspnea and cough during 4–5 min after every injection, suggesting acute hemorrhages. Eventually, the symptoms extended up to 10 – 15 min. Morphological pulmonary alterations were absent in all control rats, whereas progressive pulmonary lesions were found in all cellular structures from cocainetreated rats. The macroscopic aspects of the lungs from rats treated during 45 to 75 days with cocaine also exhibited multiple hemorrhages. Fig. 1A shows a representative control (day 60) of respiratory bronchiole with ciliated columnar epithelium, walls of collagenous fascicles of con-
115
nective tissue inserted in smooth muscle and elastic fibers surrounding the alveoli, which have arisen from adjacent ducts. Alveoli were observed as sacs with thin walls of reticular and few elastic fibers, occupied by thin capillaries. From days 7 to 15 of cocaine treatment, a decrease in the bronchiole lumen and alterations in cells from the ciliated columnar epithelium were both found. Around the walls an irregular increase of fibrous connective tissue was also found and the walls of veins and arterioles appear shrunken. Some inflammatory infiltrates (mainly macrophages and lymphocytes) were also evident at these times (not shown). At day 30 of treatment (Fig. 1B) there is evidence of hemorrhages by disruption of capillaries. The lumen of alveolar ducts, sacs and alveoli were
Fig. 1. Photomicrographs showing the effect of intraperitoneal injection of saline (A) or 30 mg/kg per day cocaine hydrochloride (B–D) on alveolar sections from rat lungs 30–75 days after administration. All sections are stained with Masson’s stain, 100 ×. Control rat at day 60 (A), showing normal alveolar ducts (ad), alveolar sacs (as), alveolus (al) and normal walls (arrows). Day 30 of cocaine treatment (B) exhibiting hemorrhage (arrows) by rupture of capillary walls. The lumen of alveolar ducts (ad), sacs and alveolus (al) are decreased by wall fibrous thickening (stars). Day 45 of cocaine treatment (C) showing alveolus lumen which appears decreased in size by intense cellular proliferation and fibrous tissue of the walls (f). Also, an increased number of macrophages with cytoplasmic granulations (arrows) can be observed. Day 75 of cocaine treatment (D) showing reduced and deformed alveoli and their interstitial space have been occupied with necrotic cells (n). The capillaries are occluded, collapsed or destroyed (arrow).
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4. Discussion
Fig. 2. Alveolar area (in mm2) of lungs from rats treated with cocaine during 75 days. Rats were daily administered i.p. either with saline (controls) or cocaine (30 mg/kg per day). Time of tissue evaluation was also 75 days. Data are expressed as mean9standard error (S.E.). * PB 0.05, Student’s t-test.
smaller and their walls appear thickened with pericapillary fibrosis and proliferation of connective tissue. In contrast with control animals, the macroscopic aspect of the lungs from rats treated during 45–75 days with cocaine exhibited multiple hemorrhages. At day 45 (Fig. 1C), the alterations of alveoli and their walls were more evident. Cellular proliferation of histiocytes and thickening of the capillary basal membrane were observed. Macrophages were found infiltrated into alveolar cavities. From days 60 to 75 of cocaine treatment, the alveoli exhibited severe abnormalities in their walls produced by numerous adhesions, intense fibroblastic activity with irregular deformities of alveoli, and altered capillaries (Fig. 1D). A significant reduction (31%) of alveolar area in lung tissue from rats after 75 days of cocaine treatment (1372.189112.66 mm2) as compared to control rats (Fig. 2) was also found (1926.27 9 20.70 mm2). At shorter times after cocaine administration (7 and 15 days) there were no effects on alveolar area as compared to control rats (data not shown).
In this work some major alterations of alveoli produced after a systemic administration of cocaine to rats are described. Several morphological alveolar changes after light microscope examination were found: intra-alveolar damage with edema, fibrosis, decreased size of alveoli and increased number of macrophages; all of them indicating that lesions are progressive and similar to those observed during human chronic abuse of cocaine (Wilkins, 1992). A significant decrease in alveolar areas from rats after 75 days of cocaine treatment as compared with controls was also observed. The general mechanism of cocaine toxicity has been summarized in four major hypothesis (Wilkins, 1992): (1) sequestration of cocaine by lungs (decreased pulmonary blood flow) and release into the blood stream; (2) cocaine-induced increased circulating levels of neurotransmitters and corticosteroids; (3) cocaine-related clinical complications (ischemic stroke, coronary ischemia and renal failure) potentiated by catecholamine actions on vascular tissues and decreased clearance of circulating catecholamines in the lungs; and (4) altered airway resistance dynamics and asthma related with catecholamine depletion. From the findings of this work and the consistent morphological changes observed over the time of exposure, different events of alveoli injury occurring during the initial phase versus the final phase of cocaine administration are suggested. During the initial phase, cocaine may produce changes of alveolar cells which in turn, may serve to explain moderate thickness of alveolar walls followed by epithelial remodeling, interstitial edema and interstitial inflammatory infiltration of granulocytes, neutrophils, eosinophils and macrophages; whereas during the final phase or ‘organizing phase’, injury is characterized by interstitial fibrosis and emphysema. It was therefore hypothesized that during the initial phase of exposure to cocaine, alveolar damage was due to dense inflammatory infiltration, while during the final phase of exposure, inflammatory infiltration is less dense and interstitial thickening is predominantly due to fibrosis.
R. Barroso-Moguel et al. / Toxicology Letters 110 (1999) 113–118
On the other hand, previous reports (Pettit and Pettit, 1994) have shown no significant differences among several routes of cocaine administration on the disposition of this drug in different tissues, suggesting that once into the body, cocaine is widely distributed, the lung being the major initial site for the drug sequestration (Philpot et al., 1977). Consequently, the findings initially support the assumption that the intraperitoneal administration of cocaine to rats represents a suitable experimental model of cocaine intoxication in humans. In addition, this model has been used in previous studies demonstrating that i.p. cocaine is also able to induce histopathological changes in kidney, testis, fetal retina and capillary tissues similar to those observed in humans (Me´ndez-Armenta et al., 1997). However, it is important to note that there are serious differences between human and animal cocaine intoxications; i.e. O’Donnell et al. (1991) have detected free silica particles in cells of the lungs from crack cocaine abusers, suggesting that this particle may, in part, account for the fibrotic changes observed during open lung biopsy. Therefore, the ethiology is though to be ischemic, secondary to chronic cocaine-induced vasoconstriction, but could also be secondary to contaminants in the inhaled cocaine. The cocaine employed in this study was free of contaminants (99% pure), as assessed by chromatographic tests performed in laboratories from the Ministry of Health (Mexico). Whether or not the morphological changes observed in human lungs are mainly due to contaminants of cocaine, remain to be elucidated. Meanwhile, the findings resemble those of human pathology. In previous findings (Me´ndez-Armenta et al., 1997), the histopathological lesions induced by cocaine were secondary to the vasoconstriction, which in turn may result in hypoxia and ischemia. This fact has been related with the demonstrated lipophilicity of cocaine, its pKa (8.5), and its chemical similarity to compounds that easily accumulate into the lungs (Wilkins, 1992). Moreover, although cocaine held in the lung is subject to some biotransformation, this is
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generally moderated (Minchin and Boyd, 1983; Bend and Serabji-Singh, 1984; Wilkins, 1992). An additional consideration for the broad spectrum of cocaine toxicity involves oxidative stress after hypoxic/ischemic events. Oxidative injury has been described as the general alteration of the redox activity in tissues, due to the action of toxic insults, with a consequent production of free radicals, which in turn are responsible for structural and functional damage of lipids, proteins and DNA (Halliwell and Gutteridge, 1985). Generation of free radicals by disruption of electron transport in mitochondria and depletion of ATP are both well-known mechanisms involved in oxidative injury. Cocaine is known to produce ATP depletion and some other markers of oxidative stress such as increased manganese-superoxide dismutase activity, decreased levels of glutathione and production of thiobarbituric acid-reactive substances (Devi and Chan, 1996; Masini et al., 1997). Moreover, cocaine has been also shown to produce parenchymal damage by its known ability to generate free radicals, leading to lipid peroxidation with consecutive damage of cytoplasmic membranes, affecting the inner membrane functional integrity (Devi and Chan, 1996, 1997), which in turn may contribute to the alveolar injury. Furthermore, it has been demostrated that some oxidative metabolites of cocaine (norcocaine and N-hydroxynorcocaine) are increased in blood and plasma of mice after systemic administration of cocaine (Benuck et al., 1989), suggesting that the oxidative metabolism of this drug may represent a considerable source of centered-oxygen species available for all tissues. Therefore, it is likely that the alterations observed in this work after cocaine administration to animals might be also due to the pro-oxidant potency which this drug can exert on lung tissue. In summary, this study shows that i.p. administration of cocaine to rats may produce severe damage to lung alveoli, including interstitial hemorrhages, thrombosis and fibrosis. These results suggest that alveolar damage is produced by a direct vasoconstrictive effect followed by a cascade of toxic events, resulting in alveolar wall thickening.
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References Anderson-Brown, T., Slotkin, T.A., Seidler, F.J., 1990. Cocaine acutely inhibits DNA synthesis in developing rat brain regions: evidence for direct actions. Brain Res. 537, 197 – 202. Bailey, M.E., Fraire, A.E., Greenbreg, S.D., Barnard, J., Cagle, P.T., 1994. Pulmonary histopathology in cocaine abusers. Hum. Pathol. 25, 203–207. Barroso-Moguel, R., Me´ndez-Armenta, M., Villeda-Herna´ndez, J., 1994. Testicular lesions by chronic administration of cocaine in rats. J. Appl. Toxicol. 14, 37–41. Barroso-Moguel, R., Me´ndez-Armenta, M., Villeda-Herna´ndez, J., 1995. Experimental nephropathy by chronic administration of cocaine in rats. Toxicology 98, 41–46. Barroso-Moguel, R., Villeda-Herna´ndez, J., Me´ndez-Armenta, M., Rı´os, C., 1997. Brain capillary lesions produced by cocaine in rats. Toxicol. Lett. 92, 9–13. Bend, J.R., Serabji-Singh, C.J., 1984. Xenobiotic metabolism by extrahepatic tissues: relationship to target organ and cell toxicity. In: Mitchel, J.R., Horning, M.G. (Eds.), Drug Metabolism and Drug Toxicity. Raven, New York, pp. 99– 136. Benuck, M., Reith, M., Sershen, H., Wiener, H., Lajtha, A., 1989. Oxidative metabolism of cocaine: comparison of brain and liver. Soc. Exp. Biol. Med. 190, 7–13. Church, W.M., Dintcheff, A.B., Grasner, K.P., 1988. Dose dependent consequences of cocaine on pregnancy outcome in the long-evans rat. Neurotoxicol. Teratol. 10, 51–58. Devi, B.G., Chan, A.W., 1996. Cocaine-induced peroxidative stress in rat liver: antioxidant enzymes and mitochondria. J. Pharmacol. Exp. Ther. 279, 359–366. Devi, B.G., Chan, A.W., 1997. Impairment of mitochondrial respiration and electron transport chain enzymes during cocaine-induced hepatic injury. Life Sci. 60, 849–855. Dow-Edwards, L.D., 1989. Long-term neurochemical and neurobehavioral consequence of cocaine-use during pregnancy. Ann. N. Y. Acad. Sci. 562, 280–289. Fleming, J.A., Byck, R., Barash, P.G., 1990. Pharmacology and therapeutic applications of cocaine. Anesthesiology 73, 518 – 531.
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Halliwell, B., Gutteridge, J.M.C., 1985. Free Radicals in Biology and Medicine. Claredon Press, Oxford. Luna, G. (Ed.), 1960. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. American Registry of Pathology, Washington DC, p. 258. Masini, A., Gallesi, D., Giovannini, F., Trenti, T., Ceccarelli, D., 1997. Membrane potential of hepatic mitochondria after acute cocaine administration in rats: the role of mitochondrial reduced glutathione. Hepathology 25, 385 – 390. Me´ndez-Armenta, M., Barroso-Moguel, R., Villeda-Herna´ndez, J., Romero-Dı´az, V., Rı´os, C., 1997. Retinal lesions in rat fetuses prenatally exposed to cocaine. Neurotoxicol. Teratol. 19, 199 – 203. Minchin, R.F., Boyd, M.R., 1983. Localization of metabolic activation and deactivation system in the lung: significance to the pulmonary toxicity of xenobiotics. Ann. Rev. Pharmacol. Toxicol. 23, 217 – 238. O’Donnell, A.D., Mappin, F.G., Sebo, T., Tazelaar, H., 1991. Interstitial pneumonitis associated with ‘crack’ cocaine abuse. Chest 100, 1155 – 1157. Pettit, H., Pettit, A.J., 1994. Disposition of cocaine in blood and brain after a single pretreatment. Brain Res. 651, 261 – 268. Philpot, R.M., Anderson, M.W., Eling, T.E., 1977. Uptake, accumulation, and metabolism of chemicals by the lung. In: Bankle and Vane (Eds.), Metabolic Functions of the Lung. Dekker, New York. Rockhold, R.W., Oden, G., Ho, I.K., Andrew, M., Farley, J.M., 1991. Glutamate receptor antagonists block cocaineinduced convulsions and death. Brain Res. Bull. 27, 721 – 723. Shimosato, K., Marley, R.J., Saito, T., 1995. Differential effects of NMDA receptor and dopamine receptor antagonists on cocaine toxicities. Pharmacol. Biochem. Behav. 51, 781 – 788. Wilkins, J.N., 1992. Brain, lung, and cardiovascular interactions with cocaine and cocaine-induced catecholamine effects. J. Addict. Dis. 11, 9 – 19.
Alveolar lesions induced by systemic administration of cocaine to rats Rosario Barroso-Moguel a,*, Juana Villeda-Herna´ndez a, Marisela Me´ndez-Armenta a, Abel Santamarı´a b, Sonia Galva´n-Arzate c a
Laboratory of Cellular Neuromorphology, National Institute of Neurology and Neurosurgery, A6. Insurgentes Sur 3877, Mexico D.F. 14269, Mexico b Laboratory of Excitatory Aminoacids, National Institute of Neurology and Neurosurgery, A6. Insurgentes Sur 3877, Mexico D.F. 14269, Mexico c Department of Neurochemistry, National Institute of Neurology and Neurosurgery, A6. Insurgentes Sur 3877, Mexico D.F. 14269, Mexico Received 6 April 1999; accepted 28 July 1999
Abstract In this work, alveolar lesions induced after systemic administration of cocaine (30 mg/kg per day, i.p.) to rats were evaluated both by light microscope analysis for morphological assessment as well as by measurement of the alveolar area as a quantitative index of the alveolar damage. Rats were examined after different times of exposure: 7, 15, 30, 45, 60 and 75 days. The histopathological evaluation of cocaine-treated rats revealed a remarkable thickening in some interalveolar septa, with interstitial hemorrhages, progressive thrombosis and transformation of reticular and elastic fibers into diffuse fibrosis. A significant decrease of the alveolar area was also observed. These findings are indicative of severe changes in capillaries, alveoli and bronchiole after cocaine exposure, which in turn may progressively disrupt the general function of the lungs. Differential mechanisms of systemic toxicity after cocaine exposure are discussed. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cocaine; Alveoli; Lungs; Bronchiole; Macrophages; Edema; Fibrosis
1. Introduction Cocaine is a well-known addictive drug exhibiting a lot of adverse effects. Cocaine and related metabolites have been shown to be responsible of * Corresponding author. Tel.: +52-5-606-3822; fax: +525-528-0095. E-mail address: [email protected] (R. Barroso-Moguel)
several toxic actions occurring in the brain, liver, kidneys and testis, among others (Fleming et al., 1990; Barroso-Moguel et al., 1994, 1995, 1997). This drug has been demonstrated to interfere with DNA synthesis (Anderson-Brown et al., 1990) and may also induce seizures and lethality associated with excitatory aminoacid and dopamine receptors in the brain (Rockhold et al., 1991; Shimosato et al., 1995). It is also known that one
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 9 9 ) 0 0 1 4 8 - 4
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of the organs with major affinity for this drug is the lung. Moreover, cocaine is also capable to induce morphological and functional changes in human lungs, such as increased number of capillaries and disrupted permeability, leading to fibrosis, hemorrhage, congestion and intra-alveolar edema, also exhibiting an inflammatory infiltrate consisting of neutrophils, lymphocytes, macrophages and eosinophils (Bailey et al., 1994). However, only little information is available concerning the morphological alterations of alveoli produced after the systemic administration of cocaine to animals. In order to provide additional information on the toxic action of cocaine as a suitable model of cocaine intoxication in humans (Barroso-Moguel et al., 1997), this study was devoted to describe and compare the morphological alveolar alterations produced by this drug at different times after its systemic administration to rats as assessed by light microscopy. Alveolar areas were also analyzed as a quantitative index of cocaineinduced lung alterations.
2. Materials and methods
2.3. Cocaine treatment Control rats received saline i.p. daily (N= 36). Treated animals (N= 36) received an aqueous solution of cocaine hydrochloride (30 mg/kg per day, i.p.) for different periods of time (7, 15, 30, 45, 60 or 75 days). Similar doses have been previously used for the study of long-term effects of cocaine in rats (Church et al., 1988; Dow-Edwards, 1989; Barroso-Moguel et al., 1994, 1995, 1997). Total amounts of cocaine received by animals were 210, 450, 900, 1350, 1800 or 2250 mg/kg during the respective periods of administration.
2.4. Morphological examination Rats from both groups were anesthetized with 3.5% chloral hydrate i.p. and then perfused transcardially at a constant pressure on days 7, 15, 30, 45, 60 and 75 of treatment. Lungs were then dissected and fixed in 10% formalin during 15 days. Lung sections were paraffin-embedded and sectioned as 4–5 mm thin slices. Hematoxilineosin and Masson’s thrichrome stains were applied (Luna, 1960). Sections were examined with a Zeiss light photomicroscope (Fomi III).
2.1. Reagents Cocaine hydrochloride (C17H22CINO4) was obtained from the Department of Psychotropic Drugs at the Ministry of Health (Mexico). Formaldehyde and staining reagents were obtained from Mallinckrodt/Baker (Mexico). All other reagents were from E. Merck (Mexico).
2.2. Animals Male Wistar bred-in-house rats (225 – 230 g) were used throughout the study. Animals were housed in acrylic box cages and provided with standard rodent Chow (Purine Chow) and water ad libitum. Animals were maintained under conditions of constant temperature (259 3°C), humidity (50 910%) and lighting (12:12 h, light:dark cycle).
2.5. Measurement of al6eolar area Alveolar areas were analysed employing a digital analysis system (Zidas; Carl Zeiss) and a micrograph amplifier (1:10). Final magnification for measurement of areas was 400×. Nine fields per section (nine sections per rat), all randomly selected, were measured and integrated. Final areas were expressed in mm2 as mean 9 standard error (S.E.).
2.6. Statistical analysis Alveolar areas from control and cocaine-treated rats were statistically compared by Student’s ttest. Data were logaritmically transformed in order to equalize the variance prior t-testing.
R. Barroso-Moguel et al. / Toxicology Letters 110 (1999) 113–118
3. Results
3.1. Morphological findings Cocaine-treated rats exhibited dyspnea and cough during 4–5 min after every injection, suggesting acute hemorrhages. Eventually, the symptoms extended up to 10 – 15 min. Morphological pulmonary alterations were absent in all control rats, whereas progressive pulmonary lesions were found in all cellular structures from cocainetreated rats. The macroscopic aspects of the lungs from rats treated during 45 to 75 days with cocaine also exhibited multiple hemorrhages. Fig. 1A shows a representative control (day 60) of respiratory bronchiole with ciliated columnar epithelium, walls of collagenous fascicles of con-
115
nective tissue inserted in smooth muscle and elastic fibers surrounding the alveoli, which have arisen from adjacent ducts. Alveoli were observed as sacs with thin walls of reticular and few elastic fibers, occupied by thin capillaries. From days 7 to 15 of cocaine treatment, a decrease in the bronchiole lumen and alterations in cells from the ciliated columnar epithelium were both found. Around the walls an irregular increase of fibrous connective tissue was also found and the walls of veins and arterioles appear shrunken. Some inflammatory infiltrates (mainly macrophages and lymphocytes) were also evident at these times (not shown). At day 30 of treatment (Fig. 1B) there is evidence of hemorrhages by disruption of capillaries. The lumen of alveolar ducts, sacs and alveoli were
Fig. 1. Photomicrographs showing the effect of intraperitoneal injection of saline (A) or 30 mg/kg per day cocaine hydrochloride (B–D) on alveolar sections from rat lungs 30–75 days after administration. All sections are stained with Masson’s stain, 100 ×. Control rat at day 60 (A), showing normal alveolar ducts (ad), alveolar sacs (as), alveolus (al) and normal walls (arrows). Day 30 of cocaine treatment (B) exhibiting hemorrhage (arrows) by rupture of capillary walls. The lumen of alveolar ducts (ad), sacs and alveolus (al) are decreased by wall fibrous thickening (stars). Day 45 of cocaine treatment (C) showing alveolus lumen which appears decreased in size by intense cellular proliferation and fibrous tissue of the walls (f). Also, an increased number of macrophages with cytoplasmic granulations (arrows) can be observed. Day 75 of cocaine treatment (D) showing reduced and deformed alveoli and their interstitial space have been occupied with necrotic cells (n). The capillaries are occluded, collapsed or destroyed (arrow).
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4. Discussion
Fig. 2. Alveolar area (in mm2) of lungs from rats treated with cocaine during 75 days. Rats were daily administered i.p. either with saline (controls) or cocaine (30 mg/kg per day). Time of tissue evaluation was also 75 days. Data are expressed as mean9standard error (S.E.). * PB 0.05, Student’s t-test.
smaller and their walls appear thickened with pericapillary fibrosis and proliferation of connective tissue. In contrast with control animals, the macroscopic aspect of the lungs from rats treated during 45–75 days with cocaine exhibited multiple hemorrhages. At day 45 (Fig. 1C), the alterations of alveoli and their walls were more evident. Cellular proliferation of histiocytes and thickening of the capillary basal membrane were observed. Macrophages were found infiltrated into alveolar cavities. From days 60 to 75 of cocaine treatment, the alveoli exhibited severe abnormalities in their walls produced by numerous adhesions, intense fibroblastic activity with irregular deformities of alveoli, and altered capillaries (Fig. 1D). A significant reduction (31%) of alveolar area in lung tissue from rats after 75 days of cocaine treatment (1372.189112.66 mm2) as compared to control rats (Fig. 2) was also found (1926.27 9 20.70 mm2). At shorter times after cocaine administration (7 and 15 days) there were no effects on alveolar area as compared to control rats (data not shown).
In this work some major alterations of alveoli produced after a systemic administration of cocaine to rats are described. Several morphological alveolar changes after light microscope examination were found: intra-alveolar damage with edema, fibrosis, decreased size of alveoli and increased number of macrophages; all of them indicating that lesions are progressive and similar to those observed during human chronic abuse of cocaine (Wilkins, 1992). A significant decrease in alveolar areas from rats after 75 days of cocaine treatment as compared with controls was also observed. The general mechanism of cocaine toxicity has been summarized in four major hypothesis (Wilkins, 1992): (1) sequestration of cocaine by lungs (decreased pulmonary blood flow) and release into the blood stream; (2) cocaine-induced increased circulating levels of neurotransmitters and corticosteroids; (3) cocaine-related clinical complications (ischemic stroke, coronary ischemia and renal failure) potentiated by catecholamine actions on vascular tissues and decreased clearance of circulating catecholamines in the lungs; and (4) altered airway resistance dynamics and asthma related with catecholamine depletion. From the findings of this work and the consistent morphological changes observed over the time of exposure, different events of alveoli injury occurring during the initial phase versus the final phase of cocaine administration are suggested. During the initial phase, cocaine may produce changes of alveolar cells which in turn, may serve to explain moderate thickness of alveolar walls followed by epithelial remodeling, interstitial edema and interstitial inflammatory infiltration of granulocytes, neutrophils, eosinophils and macrophages; whereas during the final phase or ‘organizing phase’, injury is characterized by interstitial fibrosis and emphysema. It was therefore hypothesized that during the initial phase of exposure to cocaine, alveolar damage was due to dense inflammatory infiltration, while during the final phase of exposure, inflammatory infiltration is less dense and interstitial thickening is predominantly due to fibrosis.
R. Barroso-Moguel et al. / Toxicology Letters 110 (1999) 113–118
On the other hand, previous reports (Pettit and Pettit, 1994) have shown no significant differences among several routes of cocaine administration on the disposition of this drug in different tissues, suggesting that once into the body, cocaine is widely distributed, the lung being the major initial site for the drug sequestration (Philpot et al., 1977). Consequently, the findings initially support the assumption that the intraperitoneal administration of cocaine to rats represents a suitable experimental model of cocaine intoxication in humans. In addition, this model has been used in previous studies demonstrating that i.p. cocaine is also able to induce histopathological changes in kidney, testis, fetal retina and capillary tissues similar to those observed in humans (Me´ndez-Armenta et al., 1997). However, it is important to note that there are serious differences between human and animal cocaine intoxications; i.e. O’Donnell et al. (1991) have detected free silica particles in cells of the lungs from crack cocaine abusers, suggesting that this particle may, in part, account for the fibrotic changes observed during open lung biopsy. Therefore, the ethiology is though to be ischemic, secondary to chronic cocaine-induced vasoconstriction, but could also be secondary to contaminants in the inhaled cocaine. The cocaine employed in this study was free of contaminants (99% pure), as assessed by chromatographic tests performed in laboratories from the Ministry of Health (Mexico). Whether or not the morphological changes observed in human lungs are mainly due to contaminants of cocaine, remain to be elucidated. Meanwhile, the findings resemble those of human pathology. In previous findings (Me´ndez-Armenta et al., 1997), the histopathological lesions induced by cocaine were secondary to the vasoconstriction, which in turn may result in hypoxia and ischemia. This fact has been related with the demonstrated lipophilicity of cocaine, its pKa (8.5), and its chemical similarity to compounds that easily accumulate into the lungs (Wilkins, 1992). Moreover, although cocaine held in the lung is subject to some biotransformation, this is
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generally moderated (Minchin and Boyd, 1983; Bend and Serabji-Singh, 1984; Wilkins, 1992). An additional consideration for the broad spectrum of cocaine toxicity involves oxidative stress after hypoxic/ischemic events. Oxidative injury has been described as the general alteration of the redox activity in tissues, due to the action of toxic insults, with a consequent production of free radicals, which in turn are responsible for structural and functional damage of lipids, proteins and DNA (Halliwell and Gutteridge, 1985). Generation of free radicals by disruption of electron transport in mitochondria and depletion of ATP are both well-known mechanisms involved in oxidative injury. Cocaine is known to produce ATP depletion and some other markers of oxidative stress such as increased manganese-superoxide dismutase activity, decreased levels of glutathione and production of thiobarbituric acid-reactive substances (Devi and Chan, 1996; Masini et al., 1997). Moreover, cocaine has been also shown to produce parenchymal damage by its known ability to generate free radicals, leading to lipid peroxidation with consecutive damage of cytoplasmic membranes, affecting the inner membrane functional integrity (Devi and Chan, 1996, 1997), which in turn may contribute to the alveolar injury. Furthermore, it has been demostrated that some oxidative metabolites of cocaine (norcocaine and N-hydroxynorcocaine) are increased in blood and plasma of mice after systemic administration of cocaine (Benuck et al., 1989), suggesting that the oxidative metabolism of this drug may represent a considerable source of centered-oxygen species available for all tissues. Therefore, it is likely that the alterations observed in this work after cocaine administration to animals might be also due to the pro-oxidant potency which this drug can exert on lung tissue. In summary, this study shows that i.p. administration of cocaine to rats may produce severe damage to lung alveoli, including interstitial hemorrhages, thrombosis and fibrosis. These results suggest that alveolar damage is produced by a direct vasoconstrictive effect followed by a cascade of toxic events, resulting in alveolar wall thickening.
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