- Email: [email protected]
Lung morphometry and MMP-12 expression in rats treated with intraperitoneal nicotine
Exp Toxic Pathol 2004; 55: 393–400 http://www.elsevier-deutschland.de/etp
1
Department of Histology and Embryology and 2 Department of Pharmacology and Psychobiology, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Lung morphometry and MMP-12 expression in rats treated with intraperitoneal nicotine SAMUEL SANTOS VALENÇA1, ADENILSON DE SOUZA DA FONSECA2, KATIA DA HORA1, RICARDO SANTOS2, and LUIS CRISTÓVÃO PORTO1 With 3 figures, 3 plates and 1 table Received: January 6, 2003; Revised: August 5, 2003; Accepted: August 13, 2003 Address for correspondence: LUIS CRISTÓVÃO DE MORAES SOBRINO PORTO, State University of Rio de Janeiro, Institute of Biology Roberto Alcantara Gomes, Department of Histology and Embryology, Av. Prof. Manoel de Abreu 444 3º andar, Rio de Janeiro, RJ – Brazil - 20551–170; Tel./Fax: (55 21) 2587-6511, e-mail: [email protected] Key words: Angiogenesis; metalloelastase; nicotine; lung; morphometry.
Summary
Introduction
Nicotine, a toxic tobacco component, plays an important role in the development of cardiovascular and lung diseases in smokers. Our objective was to investigate the effects of the intraperitoneal (i.p.) nicotine treatment in lung morphology. Wistar male rats (3–4 months old) were divided in five groups, a control one, and other groups treated with nicotine (1 mg/kg/day) for 8 days and sacrificed after 24, 48, 96, and 192 h. Morphometry was used to estimate the lung alveolar parenchyme and septal elastic fibers changes, and immunohistochemistry was performed to detect macrophage metalloelastase (MMP-12) and quantify vessels by immunolabelling with α-smooth muscle cells. Thickening of the alveolar septa was present in all nicotine groups, and associated with mononuclear cell infiltration, angiogenesis, and irregular areas of collapse. After 96 h, rat lungs showed macrophage, expressing MMP-12, that was also present after 192 h of recovery. Pleural and parenchyma inflammation, fibrosis and macrophage were also seen after 192 h. Intraperitoneal nicotine treated rats exhibited an increase of the volume fraction of alveolar parenchyme, a reduction of volume and surface fraction septal elastic fibers, and an increase of the numerical fraction of microvasculature vessels compared to control ones. MMP-12 was detected in groups of macrophages Wistar rats lung exhibited a progressive morphological damage after 192 hours of recovery, after 8 daily doses of 1 mg/kg body weight on i.p. nicotine.
Nicotine has major effects on respiratory, cardiovascular, and endocrinological systems, influences central neural systems, and it is also presumed to be implicated in a range of psychiatric and neurological disorders (BALFOUR and FAGERSTROM 1996). Nicotine is primarily metabolized by the liver and, to a lesser extent, by the lungs (BENOWITZ 1986; IBA et al. 1998). Nicotine has been reported to stimulate the release of pinolysosomal content of alveolar macrophages (SCHWARTZ and BOND 1972), and it is sequestered in the bronchiolar epithelium after systemic administration (SZUTS et al. 1978). Major modifications were not seen in the lung after the nicotine treatment (KIMMEL and DIAMOND 1984; ROGERS 1986). Although some evidence of adverse effects of nicotine has been found in lungs of neonates from mothers exposed to nicotine (MARITZ and WOOLWARD 1992; MARITZ and THOMAS 1995; SEKHON et al. 2002). The role of elastases in the pathogenesis of pulmonary emphysema has been the animal models focus. Attempts have been made to link cigarette smoking to the development of emphysema at chemical and cellular levels (JANOFF 1985). Both neutrophil and macrophage elastases have been implicated in chronic lung injury associated with smoking (DHAMI et al. 2000; HAUTAMAKI et al. 1997). Moreover, alveolar macrophages from the em0940-2993/04/55/05-393 $ 15.00/0
393
physematous lung produce high quantities of matrix-degrading enzymes with both elastolytic and collagenolytic activities, including metalloelastase (FINLAY 1997). Fibroplasia and deposition of extracellular matrix are partially dependent on angiogenesis and vascular remodeling. Recently, nicotine was related to angiogenesis induction (HEESCHEN et al. 2001). Despite the wide use of nicotine for smoke cessation and other pharmacological purposes, the toxic effects of its intraperitoneal (i.p.) administration remain controversial, regarding lung histology and cell response in animal models. The i.p. dose and the period of examination of the lungs were not uniform. We have studied the lung of rats with morphometry after 8 days of i.p. nicotine (1 mg/kg body weight). Moreover, the elastic fibers were also evaluated, the expression of macrophage metalloelastase (MMP-12) and the quantification of the vessel number was determined by the visualization of the αsmooth muscle actin in the vessel wall cells.
Material and methods Animals and nicotine treatment: Adult male Wistar rats (3–4 months old, body weight 250–350 g) were housed, five per cage, in a controlled room with light/dark cycle conditions (12 h light/12 h dark; lights on at 6:00 p.m.), for an acclimatization period of 3 weeks at least. Animals had free access to water and food, and the room temperature was kept around 25 ± 2 °C. Animals (n = 40) were treated with 1.0 mg/kg/day of nicotine ((–)-nicotine ([–]-1-methyl-2-[3-pyridyl]-pyrrolidine, Sigma, USA), via intraperitoneal (i.p.) injection, during 8 days. This dose corresponds to the intake range from 0.16 to 1.8 mg/kg body weight/day of habitual smokers (MARITZ and WOOLWARD 1992). Intraperitoneal nicotine treatment was accomplished in two series (2 × 20 animals) in the same conditions and with animals with the same age. The sacrifice was performed in an inhalation chamber with CO2 (FELDMAN and GUPTA 1976) with different recovery times of 24, 48, 96 and 192 h, respectively 1R, 2R, 4R, and 8R groups, with 5 animals in each group for each series. Control group (C) was manipulated under the same conditions that the experimental groups (treated with vehicle – H2O). Experimental protocol was approved by the Instituto de Biologia Roberto Alcantara Gomes – UERJ – Animal Research Committee. Histology and morphometry: In order to analyze the lung by morphometry the sample design should take care of bias related to the orientation of the structures in the lung. Estimates of areas, lengths, and numbers can be obtained for lung specimens with isotropic uniform random (IUR) tissue section (GUNDERSEN and JENSEN 1987). The IUR sections were used to avoid the influence of tissue anisotropy (WIEBE and LAURSEN 1995). After a midline thoracotomy, an intracardiac punction to collect 1.5–2 ml of blood was performed, the trachea was exposed and cannulated, and the lungs fixed by instillation of 1.8–2.0 ml of buffered formaline (10%), at a pressure of 18–22 cm H2O, during 3–4 minutes. The trachea was then ligated and the lungs, separate from the heart, were im394
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mersed in the fixative solution for 48 h. The left lung was perpendicularly sectioned to the lung base (apico-basal axis) originating two halves with portions of the upper, middle, and base of the lung. Each paraffin tissue block contained the lung halves with the sectioned portions oriented to the cutting face of the block. Serial sectioning was performed and 150 µm interval sections (IUR) were selected for staining and morphometry. Immunohistochemistry: Immunohistochemistry was used to confirm the presence and distribution of MMP-12 in lung macrophage cell populations and alpha-smooth muscle actin. To detect MMP-12, the sections (5 µm) were deparaffinized, hydrated, and after blocking of endogenous peroxidase, the sections were incubated with a goat polyclonal primary antibody MMP-12 (Santa Cruz Biotechnology, California, CA). For revelation, secondary antibody against goat IgG biotin conjugated and streptavidin complex (Santa Cruz Biotechnology) were used. Diaminobenzidine was used as chromogen, and nuclei were stained with Delafield’s hematoxylin. Negative controls were performed, replacing primary antibody by non-immuno serum and no labeling was observed. In order to visualize the vessels in the lung, the sections were treated by the same process as the above mentioned, but using a mouse monoclonal primary antibody anti-alpha-smooth muscle actin (Dako, Carpinteria, CA) and for revelation, secondary antibody against mouse IgG biotin conjugated and streptavidin complex (Dako) were used. Morphometry: To obtain uniform and proportional sampling of the parenchyma, eighteen fields (six non-overlapping fields, 150 µm apart from each of the sections) were randomly analyzed using a video-microscope (Zeissaxioplan – 20× objective lens, and JVC color video camera linked to a Sony Trinitron color video monitor), and a cycloid test-system superimposed on the monitor screen. The reference volume was estimated by point counting, using the test points hitting the parenchyma (PT). The number of points hitting the alveolar septa and the elastic fibers (PP) were counted to estimate the volume densities of these structures (V = PP /PT) (WEIBEL 1981). The number of vessels was counted to obtain the vessel numerical density (N = N/AT). Intercepts between the alveolar septa surface and intercepts between elastic fibers were counted with the cycloid arcs (IL) to estimate the surface of alveolar gas exchange and continuity of the elastic component (S = 2 · IL), respectively. A total area of 1.94 mm2 per lung parenchyma was analyzed to determine the Volume and Surface fractions of alveolar parenchyme (Vap and Sap) in sections stained with hematoxylin-eosin. The same area was examined to determine the Volume and Surface fractions of septal elastic fiber (Vse and Sse) in orcein stained sections. Vessels of microvasculature – small arteries, arterioles and venules, defined by the presence of smooth muscle cells in their wall were counted to obtain the Numerical fraction of vessels (Nv) were analyzed in immunolabelled sections with antialpha-smooth muscle actin. Vap, Sap, Vse, Sse and Nvm obtained from animals of each group were tested for normality through the Kolmogorov and Smirnov test. The control group Vap, Sap, Vse and Sse mean values were considered 100%. The difference between the volume or surface fraction in each area test in
Plate 1. Lung photomicrographs. a and b: Control rat with normal septa (arrows) and alveolar spaces (A). c: Pulmonary congestion of alveolar septa (arrows) (1R). d: Discrete pulmonary congestion, hemorrhage (H), and thickened septa (arrows) (2R). e and f: Collapsed parenchyma areas and thickened septa (arrows) and initial fibrosis (arrow heads) (4R). g: Fibrosis on Collapsed parenchyma areas (arrows head) (8R). a, c, d, e and f: ×100; b and g: ×400. Hematoxylin and eosin.
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Plate 2. Lung photomicrographs. a: Elastic fibers in control rat (arrows). b: Irregular, sparse and thickened elastic fiber in nicotine treated rat (arrows) (8R). Orcein, ×400.
1R, 2R, 4R, 8R, and the mean of the control group were obtained to calculate the ∆Vap, ∆Sap, ∆Vse and ∆Sse mean and SD. The difference between the number of vessels in each area test in 1R, 2R, 4R, 8R, and median of the control group was calculated to obtain the median, minimum and maximum values of ∆Nvm. Statistical analysis: No differences were found among the two series. One-way analysis of variance – ANOVA, followed by Tukey-Kramer multiple comparison test, using a p < 0.05 (ZAR 1999) was performed to determine the differences among ∆Vap, ∆Sap, ∆Vse and ∆Sse in 1R, 2R, 4R, 8R, and control. Kruskal-Wallis test, followed by a Dunn’s multiple comparison test, was performed to determine the differences among ∆Nvm in 1R, 2R, 4R, 8R, and control. InStat Graphpad software was used to perform statistical analysis (GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California, USA). 396
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Results Histopathology Similar results were obtained in the two experimental series. Control lungs exhibited a normal histology (plate 1a and 1b). Lung parenchyma consisted of alveoli that are connected to alveolar ducts only separated form each other by a thin alveolar septa. Capillaries containing red blood cells were evident in these thin septa. After 8 days of nicotine administration (1R) areas of enlarged alveolar septa with normal or dilated capillaries were observed (plate 1c). The examination of the lungs with two days of recovery from nicotine administration (2R) revealed the presence of alveolar septa occupied by enlarged capillaries, and foci of intra-alveolar hemorrhage (plate 1d). The vasodilatation of capillaries in the alveo-
Plate 3. Lung photomicrographs Immunostaining with α-smooth muscle actin (a–d) and MMP-12 (e–f). a: A control rat with alpha-smooth muscle actin expression on the smooth muscle cells of the vessel walls (arrows). b, c, and d: treated groups with nicotine showing an increase of vessel numbers with alpha-smooth muscle actin expression (arrows). e: A control rat lung. MMP-12 is not expressed. f, g, and h: treated groups with nicotine showing an increase of MMP-12 expression (arrows and red circle). DAB and Delafield’s hematoxilin, ×400.
lar septa and the collapse of the architecture of the lungs were evident after 8 days of nicotine treatment with 96 hours and 192 hours of recovery (4R – plate 1e, 8R – plate 1f).
Pleura and parenchyma collapse and condensation were observed in animals of the 8R group; intermingled with fibroblasts collagen fibers and mononuclear inflammatory cells were seen in these areas, the vessels were Exp Toxic Pathol 55 (2004) 5
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Table 1. Morphometric data of rats treated with i.p. nicotine during 8 days and sacrificed with 1 (1R), 2 (2R) , 4 (4R) and 8 (8R) days of recovery. Groups
Control
1R
2R
4R
8R
∆Vap ∆Vse ∆Sap ∆Sse ∆Nvp
100.0 ± 11.4 100.0 ± 30.0 100.0 ± 16.9 100.0 ± 15.2 0 (0–6)
97.3 ± 15.6 88.3 ± 28.1 69.3 ± 17.2 98.1 ± 12.1 3 (0–15)
111.4 ± 20.3 102.2 ± 44.4 53.4 ± 19.1 105.1 ± 18.7 3 (0–15)
122.0 ± 18.0 38.9 ± 23.1 44.2 ± 21.1 60.5 ± 21.6 3 (0–9)
126.9 ± 23.5 33.0 ± 23.5 32.4 ± 17.1 49.1 ± 16.9 9 (3–24)
Values are reported as mean ± Standard Deviation for 5 animals for ∆Vap (volume fraction of alveolar parenchyme), ∆Vse (volume fraction of septal elastic fiber), ∆Sap surface fraction of alveolar parenchyme), and ∆Sse (surface fraction of septal elastic fiber), as the difference of percentage from mean in the control group in 18 random, non-coincident fields per rat), and median, minimum and maximum values for ∆Nvp(numerical fraction of parenchymal vessels).
Fig. 1. Difference of septal elastic fibers and alveolar parenchyme volume fractions of intraperitoneal nicotine treated rats after 24, 48, 96, and 192 h of recovery time, respectively, groups 1R, 2R, 4R, and 8R from the control (100%). Statistical significance determined by ANOVA, followed by Tukey-Kramer multiple comparison test.
Fig. 2. Difference of septal elastic fibers, and alveolar parenchyme surface fractions of intraperitoneal nicotine treated rats after 24, 48, 96, and 192 h of recovery time, respectively, groups 1R, 2R, 4R, and 8R from the control (100%). Statistical significance determined by ANOVA, followed by Tukey-Kramer multiple comparison test.
dilated, and many capillaries were also found in these regions (plate 1g). Elastin in the alveolar septa was observed as branching delicate fibers in the control group (plate 2a). The treated and recovered groups (2R and 4R) showed patterns corresponding to an initial elastolysis. Elastic deposits with a fragmented and irregular aspect were seen in the lungs of 8R group (plate 2b). Normal lung small vessels of control (plate 3a) and i.p. nicotine treated groups (plates 3b, 3c, and 3d) were shown by immunohistochemistry with an alpha-smooth muscle actin antibody. The nicotine treatment increased the number of these vessels, mainly in 4R (plate 3b) and 8R groups (Pplates 3c and 3d).
In the control group, there was not MMP-12 activity observed in macrophages (plate 3e). Only macrophages located in peribronchiolar infiltrates expressed MMP-12 in 4R group (plates 3f and 3g). Both alveolar and septal macrophages in 8R group overexpressed MMP-12 (plate 3h).
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Morphometry Table 1 summarizes the differences of ∆Vap, ∆Sap, ∆Vse, ∆Sse and ∆Nvm in 1R, 2R, 4R, and 8R groups. An increase in the volume fraction of alveolar parenchyme (∆Vap) was not accompanied by a proportional growth in the septal elastic fibers (∆Vse). These re-
Fig. 3. Difference of the numerical fraction of parenchyma vessels of intraperitoneal nicotine treated rats after 24, 48, 96, and 192 h of recovery time, respectively, groups 1R, 2R, 4R, and 8R from the median of control. Statistical significance determined by Kruskal-Wallis followed by a Dunn’s multiple comparison test.
sults are illustrated in figure 1. The alveolar parenchyme occupied more 11%, 22%, and 26% of control lung volume (respectively, 2R, 4R, and 8R, p < 0.001) and a Vse 61% decrease difference in 4R, and 67% in 8R groups (p < 0.001) were detected when compared to control group. When differences in the surface fraction of the alveolar parenchyme among the recovery time and the control groups (fig. 2) were analyzed, a progressive reduction of this functional surface was observed. A 31% reduction was already evident in 1R (p < 0.001). The surface fraction of elastic fiber at the septa was also decreased in parallel with the decrease found in the ∆Vse of both 4R and 8R groups (40% and 51% respectively, p < 0.001) An increase of ∆Nvp (fig. 3) was already observed in the 1R group (median = 3, p < 0.01), and a marked modification in the pattern of microvasculature vessels was found in the 8R group (median = 9, p < 0.001).
Discussion Currently, nicotine replacement therapy is used to assist in tobacco cessation by the use of brief transdermal patches, chewing gum, nasal spray, or inhaler therapy (BALFOUR and FAGERSTROM 1996; ZEVIN et al. 1998), but few studies have investigated the lung morphology after i.p. nicotine administration. Our results demonstrate an important alteration in the pulmonary structure caused by 8 days of nicotine (1 mg/kg/day) administration during the first 8 days of recovery. The dose administered was compatible with
previous studies (ROGERS 1986; MARITZ 1988; SEKHON et al. 2002). In our experiment, administration of nicotine causes a disorganization of lung parenchyma, reduction in the Sap, Sse and Vse, an increase of Vsp and MMP12 expression in macrophages in 4R and 8R groups. These results differ from KIMMEL and DIAMOND (1984), who did not observe modifications of the lung tissue volume density. This could be explained by a cumulative effect of nicotine in our experiment, as they administered 3 mg/kg or 7.5 mg/kg a single time and lungs analyzed after 4 weeks. ROGERS et al. (1986) submitted rats to i.p. nicotine and reported the reduction of bronchioli secretory cell numbers at nicotine plasma concentrations of 650 ng/ml (obtained with subcutaneous doses of 10 mg/kg 2 times a day) and increased number to 2360 ng/ml (obtained with i.p. dose of 7.5 mg/kg). In our model, nicotine was daily administered, although in a smaller amount, and lung morphometry was performed only up to 8 days after treatment. Although we cannot exclude the reorganization of the lung morphology when maintaining rats for a long period of recovery without nicotine administration, lesions were more significant after 8 days instead of 4 days of recovery. Our results showed some lung modifications after the treatment with nicotine that bring about changes in the architecture affecting elastic fibers, alveolar septa, and the expression of MMP-12 in macrophages. Initially, the alveolar septa thickening may be related to modifications in the vascular bed, causing inflammatory infiltration and edema. We also detected many mononuclear inflammatory cells in these areas. Morphometry was performed to investigate the vessels in the inflammatory areas of these lungs. Data show an increase of numerical fraction of vessels in the treated group. These results in relation to the control group suggest a process of early angiogenesis in rat lungs. Moreover, during the experiment reduction of the respiratory surface area was concomitant to the elastic fibers disorganization of the alveolar septa, despite the nicotine administration interruption on day 8. Animals also exhibited collapse and condensation in the parenchyma and pleura with a morphological fibrogenic process. HEESCHEN et al. (2001) showed that in the smoking absence, nicotine stimulates angiogenesis causing inflammation, ischemia, atherosclerosis, and cancer in murine models. Nicotine concentration as found in the plasma of smokers, stimulates DNA synthesis and proliferation of endothelial cells in vitro via nicotinic acetylcholine receptors (nAChR) (VILLABLANCA 1998). MMP12 expression in macrophages may digest structural components of the septa outside the cells, facilitating the collapse and the congestion of the lung parenchyma with the reduction of Vse and Sse. Although the quantity of intraperitoneal nicotine administered is compatible with usual human smoking habits, the manifestations of the toxicity reported in this work should be subject of additional experiments. However, nicotine has been shown to promote the release of fibroblast growth factor (bFGF) and endothelin from endothelial cells (HEESCHEN et al. Exp Toxic Pathol 55 (2004) 5
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2001; LEE and WRIGHT 1999; CONKLIN et al. 2002), to enhance the proliferation of smooth-muscle cells in culture by inducing the bFGF and TGF-β1 secretion, and to increase the production of matrix metalloproteinases (CARTY et al. 1996) that altogether facilitate angiogenesis (SCHWARTZ 2002). In conclusion, rats submitted to i.p. nicotine administration during 8 days with recovery time exhibit pulmonary interstitial edema and thickened alveolar septa that leads to a decrease of surface gas exchange, mononuclear inflammatory infiltration with MMP-12 expression inducing elastic fibers degradation and collapse of parenchyma associated to angiogenesis processes. Acknowledgment: The authors wish to thank Dr. ANDRÉA MONTE ALTO COSTA and Dr. CARLOS ALBERTO MANDARIM-DE-LACERDA for their comments, suggestions, and precious advice on morphometry. Supported by: FAPERJ, CNPQ, CAPES, CEJAG and UERJ.
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induced emphysema in mice. Science 1997; 277: 2002–2004. HEESCHEN C, JANG JJ, WEIS M, et al.: Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med 2001; 7: 833–839. IBA MM, SCHOLL H, FUNG J, et al.: Induction of pulmonary CYP1A1 by nicotine. Xenobiotica 1998; 28: 827–843. JANOFF A: Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis 1985; 132: 417–433. KIMMEL EC, DIAMOND L: The role of nicotine in the pathogenesis of pulmonary emphysema. Am Rev Respir Dis 1984; 129: 112–117. LEE WO, WRIGHT SM: Production of endothelin by cultured human endothelial cells following exposure to nicotine or caffeine. Metabolism 1999; 48: 845–848. MARITZ GS: Effect of maternal nicotine exposure on growth in vivo of lung tissue of neonatal rats. Biol Neonate 1988; 53: 163–170. MARITZ GS, THOMAS RA: Maternal nicotine exposure: response of type II pneumocytes of neonatal rat pups. Cell Biol Int 1995; 19: 323–331. MARITZ GS, WOOLWARD K: Effect of maternal nicotine exposure on neonatal lung elastic tissue and possible consequences. S Afr Med J 1992; 81: 517–519. ROGERS DF, WILLIAMS DA, JEFFERY PK: Nicotine does not cause ‘bronchitis’ in the rat. Clin Sci (Lond) 1986; 70: 427–433. SCHWARTZ MA: Nicotine-induced angiogenesis. J Clin Psychiatry 2002; 63: 949–950. SCHWARTZ SL, BOND JC: Stimulation of release of pinolysosomal contents of macrophages by nicotine. J Pharmacol Exp Ther 1972; 183: 378–384. SEKHON HS, KELLER JA, PROSKOCIL BJ, et al.: Maternal nicotine exposure upregulates collagen gene expression in fetal monkey lung. Association with alpha7 nicotinic acetylcholine receptors. Am J Respir Cell Mol Biol 2002; 26: 31–41. SZUTS T, OLSSON S, LINDQUIST NG, et al.: Long-term fate of [14C]nicotine in the mouse: retention in the bronchi, melanin-containing tissues and urinary bladder wall. Toxicology 1978; 10: 207–220. VILLABLANCA AC: Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Physiol 1998; 84: 2089–2098. WEIBEL ER: Stereological methods in cell biology: where are we – where are we going? J Histochem Cytochem 1981; 29: 1043–1052. WIEBE BM, LAURSEN H: Human lung volume, alveolar surface area, and capillary length. Microsc Res Tech 1995; 32: 255–262. ZEVIN S, JACOB P, III, BENOWITZ NL: Dose-related cardiovascular and endocrine effects of transdermal nicotine. Clin Pharmacol Ther 1998; 64: 87–95.
1
Department of Histology and Embryology and 2 Department of Pharmacology and Psychobiology, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Lung morphometry and MMP-12 expression in rats treated with intraperitoneal nicotine SAMUEL SANTOS VALENÇA1, ADENILSON DE SOUZA DA FONSECA2, KATIA DA HORA1, RICARDO SANTOS2, and LUIS CRISTÓVÃO PORTO1 With 3 figures, 3 plates and 1 table Received: January 6, 2003; Revised: August 5, 2003; Accepted: August 13, 2003 Address for correspondence: LUIS CRISTÓVÃO DE MORAES SOBRINO PORTO, State University of Rio de Janeiro, Institute of Biology Roberto Alcantara Gomes, Department of Histology and Embryology, Av. Prof. Manoel de Abreu 444 3º andar, Rio de Janeiro, RJ – Brazil - 20551–170; Tel./Fax: (55 21) 2587-6511, e-mail: [email protected] Key words: Angiogenesis; metalloelastase; nicotine; lung; morphometry.
Summary
Introduction
Nicotine, a toxic tobacco component, plays an important role in the development of cardiovascular and lung diseases in smokers. Our objective was to investigate the effects of the intraperitoneal (i.p.) nicotine treatment in lung morphology. Wistar male rats (3–4 months old) were divided in five groups, a control one, and other groups treated with nicotine (1 mg/kg/day) for 8 days and sacrificed after 24, 48, 96, and 192 h. Morphometry was used to estimate the lung alveolar parenchyme and septal elastic fibers changes, and immunohistochemistry was performed to detect macrophage metalloelastase (MMP-12) and quantify vessels by immunolabelling with α-smooth muscle cells. Thickening of the alveolar septa was present in all nicotine groups, and associated with mononuclear cell infiltration, angiogenesis, and irregular areas of collapse. After 96 h, rat lungs showed macrophage, expressing MMP-12, that was also present after 192 h of recovery. Pleural and parenchyma inflammation, fibrosis and macrophage were also seen after 192 h. Intraperitoneal nicotine treated rats exhibited an increase of the volume fraction of alveolar parenchyme, a reduction of volume and surface fraction septal elastic fibers, and an increase of the numerical fraction of microvasculature vessels compared to control ones. MMP-12 was detected in groups of macrophages Wistar rats lung exhibited a progressive morphological damage after 192 hours of recovery, after 8 daily doses of 1 mg/kg body weight on i.p. nicotine.
Nicotine has major effects on respiratory, cardiovascular, and endocrinological systems, influences central neural systems, and it is also presumed to be implicated in a range of psychiatric and neurological disorders (BALFOUR and FAGERSTROM 1996). Nicotine is primarily metabolized by the liver and, to a lesser extent, by the lungs (BENOWITZ 1986; IBA et al. 1998). Nicotine has been reported to stimulate the release of pinolysosomal content of alveolar macrophages (SCHWARTZ and BOND 1972), and it is sequestered in the bronchiolar epithelium after systemic administration (SZUTS et al. 1978). Major modifications were not seen in the lung after the nicotine treatment (KIMMEL and DIAMOND 1984; ROGERS 1986). Although some evidence of adverse effects of nicotine has been found in lungs of neonates from mothers exposed to nicotine (MARITZ and WOOLWARD 1992; MARITZ and THOMAS 1995; SEKHON et al. 2002). The role of elastases in the pathogenesis of pulmonary emphysema has been the animal models focus. Attempts have been made to link cigarette smoking to the development of emphysema at chemical and cellular levels (JANOFF 1985). Both neutrophil and macrophage elastases have been implicated in chronic lung injury associated with smoking (DHAMI et al. 2000; HAUTAMAKI et al. 1997). Moreover, alveolar macrophages from the em0940-2993/04/55/05-393 $ 15.00/0
393
physematous lung produce high quantities of matrix-degrading enzymes with both elastolytic and collagenolytic activities, including metalloelastase (FINLAY 1997). Fibroplasia and deposition of extracellular matrix are partially dependent on angiogenesis and vascular remodeling. Recently, nicotine was related to angiogenesis induction (HEESCHEN et al. 2001). Despite the wide use of nicotine for smoke cessation and other pharmacological purposes, the toxic effects of its intraperitoneal (i.p.) administration remain controversial, regarding lung histology and cell response in animal models. The i.p. dose and the period of examination of the lungs were not uniform. We have studied the lung of rats with morphometry after 8 days of i.p. nicotine (1 mg/kg body weight). Moreover, the elastic fibers were also evaluated, the expression of macrophage metalloelastase (MMP-12) and the quantification of the vessel number was determined by the visualization of the αsmooth muscle actin in the vessel wall cells.
Material and methods Animals and nicotine treatment: Adult male Wistar rats (3–4 months old, body weight 250–350 g) were housed, five per cage, in a controlled room with light/dark cycle conditions (12 h light/12 h dark; lights on at 6:00 p.m.), for an acclimatization period of 3 weeks at least. Animals had free access to water and food, and the room temperature was kept around 25 ± 2 °C. Animals (n = 40) were treated with 1.0 mg/kg/day of nicotine ((–)-nicotine ([–]-1-methyl-2-[3-pyridyl]-pyrrolidine, Sigma, USA), via intraperitoneal (i.p.) injection, during 8 days. This dose corresponds to the intake range from 0.16 to 1.8 mg/kg body weight/day of habitual smokers (MARITZ and WOOLWARD 1992). Intraperitoneal nicotine treatment was accomplished in two series (2 × 20 animals) in the same conditions and with animals with the same age. The sacrifice was performed in an inhalation chamber with CO2 (FELDMAN and GUPTA 1976) with different recovery times of 24, 48, 96 and 192 h, respectively 1R, 2R, 4R, and 8R groups, with 5 animals in each group for each series. Control group (C) was manipulated under the same conditions that the experimental groups (treated with vehicle – H2O). Experimental protocol was approved by the Instituto de Biologia Roberto Alcantara Gomes – UERJ – Animal Research Committee. Histology and morphometry: In order to analyze the lung by morphometry the sample design should take care of bias related to the orientation of the structures in the lung. Estimates of areas, lengths, and numbers can be obtained for lung specimens with isotropic uniform random (IUR) tissue section (GUNDERSEN and JENSEN 1987). The IUR sections were used to avoid the influence of tissue anisotropy (WIEBE and LAURSEN 1995). After a midline thoracotomy, an intracardiac punction to collect 1.5–2 ml of blood was performed, the trachea was exposed and cannulated, and the lungs fixed by instillation of 1.8–2.0 ml of buffered formaline (10%), at a pressure of 18–22 cm H2O, during 3–4 minutes. The trachea was then ligated and the lungs, separate from the heart, were im394
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mersed in the fixative solution for 48 h. The left lung was perpendicularly sectioned to the lung base (apico-basal axis) originating two halves with portions of the upper, middle, and base of the lung. Each paraffin tissue block contained the lung halves with the sectioned portions oriented to the cutting face of the block. Serial sectioning was performed and 150 µm interval sections (IUR) were selected for staining and morphometry. Immunohistochemistry: Immunohistochemistry was used to confirm the presence and distribution of MMP-12 in lung macrophage cell populations and alpha-smooth muscle actin. To detect MMP-12, the sections (5 µm) were deparaffinized, hydrated, and after blocking of endogenous peroxidase, the sections were incubated with a goat polyclonal primary antibody MMP-12 (Santa Cruz Biotechnology, California, CA). For revelation, secondary antibody against goat IgG biotin conjugated and streptavidin complex (Santa Cruz Biotechnology) were used. Diaminobenzidine was used as chromogen, and nuclei were stained with Delafield’s hematoxylin. Negative controls were performed, replacing primary antibody by non-immuno serum and no labeling was observed. In order to visualize the vessels in the lung, the sections were treated by the same process as the above mentioned, but using a mouse monoclonal primary antibody anti-alpha-smooth muscle actin (Dako, Carpinteria, CA) and for revelation, secondary antibody against mouse IgG biotin conjugated and streptavidin complex (Dako) were used. Morphometry: To obtain uniform and proportional sampling of the parenchyma, eighteen fields (six non-overlapping fields, 150 µm apart from each of the sections) were randomly analyzed using a video-microscope (Zeissaxioplan – 20× objective lens, and JVC color video camera linked to a Sony Trinitron color video monitor), and a cycloid test-system superimposed on the monitor screen. The reference volume was estimated by point counting, using the test points hitting the parenchyma (PT). The number of points hitting the alveolar septa and the elastic fibers (PP) were counted to estimate the volume densities of these structures (V = PP /PT) (WEIBEL 1981). The number of vessels was counted to obtain the vessel numerical density (N = N/AT). Intercepts between the alveolar septa surface and intercepts between elastic fibers were counted with the cycloid arcs (IL) to estimate the surface of alveolar gas exchange and continuity of the elastic component (S = 2 · IL), respectively. A total area of 1.94 mm2 per lung parenchyma was analyzed to determine the Volume and Surface fractions of alveolar parenchyme (Vap and Sap) in sections stained with hematoxylin-eosin. The same area was examined to determine the Volume and Surface fractions of septal elastic fiber (Vse and Sse) in orcein stained sections. Vessels of microvasculature – small arteries, arterioles and venules, defined by the presence of smooth muscle cells in their wall were counted to obtain the Numerical fraction of vessels (Nv) were analyzed in immunolabelled sections with antialpha-smooth muscle actin. Vap, Sap, Vse, Sse and Nvm obtained from animals of each group were tested for normality through the Kolmogorov and Smirnov test. The control group Vap, Sap, Vse and Sse mean values were considered 100%. The difference between the volume or surface fraction in each area test in
Plate 1. Lung photomicrographs. a and b: Control rat with normal septa (arrows) and alveolar spaces (A). c: Pulmonary congestion of alveolar septa (arrows) (1R). d: Discrete pulmonary congestion, hemorrhage (H), and thickened septa (arrows) (2R). e and f: Collapsed parenchyma areas and thickened septa (arrows) and initial fibrosis (arrow heads) (4R). g: Fibrosis on Collapsed parenchyma areas (arrows head) (8R). a, c, d, e and f: ×100; b and g: ×400. Hematoxylin and eosin.
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Plate 2. Lung photomicrographs. a: Elastic fibers in control rat (arrows). b: Irregular, sparse and thickened elastic fiber in nicotine treated rat (arrows) (8R). Orcein, ×400.
1R, 2R, 4R, 8R, and the mean of the control group were obtained to calculate the ∆Vap, ∆Sap, ∆Vse and ∆Sse mean and SD. The difference between the number of vessels in each area test in 1R, 2R, 4R, 8R, and median of the control group was calculated to obtain the median, minimum and maximum values of ∆Nvm. Statistical analysis: No differences were found among the two series. One-way analysis of variance – ANOVA, followed by Tukey-Kramer multiple comparison test, using a p < 0.05 (ZAR 1999) was performed to determine the differences among ∆Vap, ∆Sap, ∆Vse and ∆Sse in 1R, 2R, 4R, 8R, and control. Kruskal-Wallis test, followed by a Dunn’s multiple comparison test, was performed to determine the differences among ∆Nvm in 1R, 2R, 4R, 8R, and control. InStat Graphpad software was used to perform statistical analysis (GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California, USA). 396
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Results Histopathology Similar results were obtained in the two experimental series. Control lungs exhibited a normal histology (plate 1a and 1b). Lung parenchyma consisted of alveoli that are connected to alveolar ducts only separated form each other by a thin alveolar septa. Capillaries containing red blood cells were evident in these thin septa. After 8 days of nicotine administration (1R) areas of enlarged alveolar septa with normal or dilated capillaries were observed (plate 1c). The examination of the lungs with two days of recovery from nicotine administration (2R) revealed the presence of alveolar septa occupied by enlarged capillaries, and foci of intra-alveolar hemorrhage (plate 1d). The vasodilatation of capillaries in the alveo-
Plate 3. Lung photomicrographs Immunostaining with α-smooth muscle actin (a–d) and MMP-12 (e–f). a: A control rat with alpha-smooth muscle actin expression on the smooth muscle cells of the vessel walls (arrows). b, c, and d: treated groups with nicotine showing an increase of vessel numbers with alpha-smooth muscle actin expression (arrows). e: A control rat lung. MMP-12 is not expressed. f, g, and h: treated groups with nicotine showing an increase of MMP-12 expression (arrows and red circle). DAB and Delafield’s hematoxilin, ×400.
lar septa and the collapse of the architecture of the lungs were evident after 8 days of nicotine treatment with 96 hours and 192 hours of recovery (4R – plate 1e, 8R – plate 1f).
Pleura and parenchyma collapse and condensation were observed in animals of the 8R group; intermingled with fibroblasts collagen fibers and mononuclear inflammatory cells were seen in these areas, the vessels were Exp Toxic Pathol 55 (2004) 5
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Table 1. Morphometric data of rats treated with i.p. nicotine during 8 days and sacrificed with 1 (1R), 2 (2R) , 4 (4R) and 8 (8R) days of recovery. Groups
Control
1R
2R
4R
8R
∆Vap ∆Vse ∆Sap ∆Sse ∆Nvp
100.0 ± 11.4 100.0 ± 30.0 100.0 ± 16.9 100.0 ± 15.2 0 (0–6)
97.3 ± 15.6 88.3 ± 28.1 69.3 ± 17.2 98.1 ± 12.1 3 (0–15)
111.4 ± 20.3 102.2 ± 44.4 53.4 ± 19.1 105.1 ± 18.7 3 (0–15)
122.0 ± 18.0 38.9 ± 23.1 44.2 ± 21.1 60.5 ± 21.6 3 (0–9)
126.9 ± 23.5 33.0 ± 23.5 32.4 ± 17.1 49.1 ± 16.9 9 (3–24)
Values are reported as mean ± Standard Deviation for 5 animals for ∆Vap (volume fraction of alveolar parenchyme), ∆Vse (volume fraction of septal elastic fiber), ∆Sap surface fraction of alveolar parenchyme), and ∆Sse (surface fraction of septal elastic fiber), as the difference of percentage from mean in the control group in 18 random, non-coincident fields per rat), and median, minimum and maximum values for ∆Nvp(numerical fraction of parenchymal vessels).
Fig. 1. Difference of septal elastic fibers and alveolar parenchyme volume fractions of intraperitoneal nicotine treated rats after 24, 48, 96, and 192 h of recovery time, respectively, groups 1R, 2R, 4R, and 8R from the control (100%). Statistical significance determined by ANOVA, followed by Tukey-Kramer multiple comparison test.
Fig. 2. Difference of septal elastic fibers, and alveolar parenchyme surface fractions of intraperitoneal nicotine treated rats after 24, 48, 96, and 192 h of recovery time, respectively, groups 1R, 2R, 4R, and 8R from the control (100%). Statistical significance determined by ANOVA, followed by Tukey-Kramer multiple comparison test.
dilated, and many capillaries were also found in these regions (plate 1g). Elastin in the alveolar septa was observed as branching delicate fibers in the control group (plate 2a). The treated and recovered groups (2R and 4R) showed patterns corresponding to an initial elastolysis. Elastic deposits with a fragmented and irregular aspect were seen in the lungs of 8R group (plate 2b). Normal lung small vessels of control (plate 3a) and i.p. nicotine treated groups (plates 3b, 3c, and 3d) were shown by immunohistochemistry with an alpha-smooth muscle actin antibody. The nicotine treatment increased the number of these vessels, mainly in 4R (plate 3b) and 8R groups (Pplates 3c and 3d).
In the control group, there was not MMP-12 activity observed in macrophages (plate 3e). Only macrophages located in peribronchiolar infiltrates expressed MMP-12 in 4R group (plates 3f and 3g). Both alveolar and septal macrophages in 8R group overexpressed MMP-12 (plate 3h).
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Morphometry Table 1 summarizes the differences of ∆Vap, ∆Sap, ∆Vse, ∆Sse and ∆Nvm in 1R, 2R, 4R, and 8R groups. An increase in the volume fraction of alveolar parenchyme (∆Vap) was not accompanied by a proportional growth in the septal elastic fibers (∆Vse). These re-
Fig. 3. Difference of the numerical fraction of parenchyma vessels of intraperitoneal nicotine treated rats after 24, 48, 96, and 192 h of recovery time, respectively, groups 1R, 2R, 4R, and 8R from the median of control. Statistical significance determined by Kruskal-Wallis followed by a Dunn’s multiple comparison test.
sults are illustrated in figure 1. The alveolar parenchyme occupied more 11%, 22%, and 26% of control lung volume (respectively, 2R, 4R, and 8R, p < 0.001) and a Vse 61% decrease difference in 4R, and 67% in 8R groups (p < 0.001) were detected when compared to control group. When differences in the surface fraction of the alveolar parenchyme among the recovery time and the control groups (fig. 2) were analyzed, a progressive reduction of this functional surface was observed. A 31% reduction was already evident in 1R (p < 0.001). The surface fraction of elastic fiber at the septa was also decreased in parallel with the decrease found in the ∆Vse of both 4R and 8R groups (40% and 51% respectively, p < 0.001) An increase of ∆Nvp (fig. 3) was already observed in the 1R group (median = 3, p < 0.01), and a marked modification in the pattern of microvasculature vessels was found in the 8R group (median = 9, p < 0.001).
Discussion Currently, nicotine replacement therapy is used to assist in tobacco cessation by the use of brief transdermal patches, chewing gum, nasal spray, or inhaler therapy (BALFOUR and FAGERSTROM 1996; ZEVIN et al. 1998), but few studies have investigated the lung morphology after i.p. nicotine administration. Our results demonstrate an important alteration in the pulmonary structure caused by 8 days of nicotine (1 mg/kg/day) administration during the first 8 days of recovery. The dose administered was compatible with
previous studies (ROGERS 1986; MARITZ 1988; SEKHON et al. 2002). In our experiment, administration of nicotine causes a disorganization of lung parenchyma, reduction in the Sap, Sse and Vse, an increase of Vsp and MMP12 expression in macrophages in 4R and 8R groups. These results differ from KIMMEL and DIAMOND (1984), who did not observe modifications of the lung tissue volume density. This could be explained by a cumulative effect of nicotine in our experiment, as they administered 3 mg/kg or 7.5 mg/kg a single time and lungs analyzed after 4 weeks. ROGERS et al. (1986) submitted rats to i.p. nicotine and reported the reduction of bronchioli secretory cell numbers at nicotine plasma concentrations of 650 ng/ml (obtained with subcutaneous doses of 10 mg/kg 2 times a day) and increased number to 2360 ng/ml (obtained with i.p. dose of 7.5 mg/kg). In our model, nicotine was daily administered, although in a smaller amount, and lung morphometry was performed only up to 8 days after treatment. Although we cannot exclude the reorganization of the lung morphology when maintaining rats for a long period of recovery without nicotine administration, lesions were more significant after 8 days instead of 4 days of recovery. Our results showed some lung modifications after the treatment with nicotine that bring about changes in the architecture affecting elastic fibers, alveolar septa, and the expression of MMP-12 in macrophages. Initially, the alveolar septa thickening may be related to modifications in the vascular bed, causing inflammatory infiltration and edema. We also detected many mononuclear inflammatory cells in these areas. Morphometry was performed to investigate the vessels in the inflammatory areas of these lungs. Data show an increase of numerical fraction of vessels in the treated group. These results in relation to the control group suggest a process of early angiogenesis in rat lungs. Moreover, during the experiment reduction of the respiratory surface area was concomitant to the elastic fibers disorganization of the alveolar septa, despite the nicotine administration interruption on day 8. Animals also exhibited collapse and condensation in the parenchyma and pleura with a morphological fibrogenic process. HEESCHEN et al. (2001) showed that in the smoking absence, nicotine stimulates angiogenesis causing inflammation, ischemia, atherosclerosis, and cancer in murine models. Nicotine concentration as found in the plasma of smokers, stimulates DNA synthesis and proliferation of endothelial cells in vitro via nicotinic acetylcholine receptors (nAChR) (VILLABLANCA 1998). MMP12 expression in macrophages may digest structural components of the septa outside the cells, facilitating the collapse and the congestion of the lung parenchyma with the reduction of Vse and Sse. Although the quantity of intraperitoneal nicotine administered is compatible with usual human smoking habits, the manifestations of the toxicity reported in this work should be subject of additional experiments. However, nicotine has been shown to promote the release of fibroblast growth factor (bFGF) and endothelin from endothelial cells (HEESCHEN et al. Exp Toxic Pathol 55 (2004) 5
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2001; LEE and WRIGHT 1999; CONKLIN et al. 2002), to enhance the proliferation of smooth-muscle cells in culture by inducing the bFGF and TGF-β1 secretion, and to increase the production of matrix metalloproteinases (CARTY et al. 1996) that altogether facilitate angiogenesis (SCHWARTZ 2002). In conclusion, rats submitted to i.p. nicotine administration during 8 days with recovery time exhibit pulmonary interstitial edema and thickened alveolar septa that leads to a decrease of surface gas exchange, mononuclear inflammatory infiltration with MMP-12 expression inducing elastic fibers degradation and collapse of parenchyma associated to angiogenesis processes. Acknowledgment: The authors wish to thank Dr. ANDRÉA MONTE ALTO COSTA and Dr. CARLOS ALBERTO MANDARIM-DE-LACERDA for their comments, suggestions, and precious advice on morphometry. Supported by: FAPERJ, CNPQ, CAPES, CEJAG and UERJ.
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