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Depts of 1 Pharmacology, 2 Pathology and 4 Physiology, University of València, 3 Dept of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigacions Cientificas, Barcelona, Spain, Zambon Group Spa, 5 Bresso, Italy and 6 , Barcelona, Spain
CORRESPONDENCE: J. Morcillo, Dept of Pharmacology, Faculty of Medicine, Av. Blasco Ibanez 15, E-46010, Valencia, Spain. Fax: 34 963864622
Keywords: bleomycin, inflammation, N-acetylcysteine, pulmonary fibrosis, rat
Received: June 5, 2000
Accepted January 25, 2001
The present work was supported by grant 1FD97-1143 from the European Union (Regional Development Funds; FEDER), CICYT (Spanish Government) and Regional Government (Generalitat Valenciana), and a research grant from Zambon Group (Milano, Italy and Barcelona, Spain).
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Abstract |
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NAC (3 mmol·kg–1; oral) was given daily from 1 week prior to a single intratracheal instillation of bleomycin (2.5 U·kg–1) or saline, until 14 days postinstillation.
NAC partially decreased the augmented collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,354±386 and 3,416±326 µg·lung–1 in vehicle-treated and NAC-treated rats, respectively; p<0.05). The histological assessment using a semiquantitative score showed less collagen deposition and inflammatory cells in NAC-treated rats compared to those receiving bleomycin alone. NAC failed to inhibit the bleomycin-induced increases in lung wet weight and in cell counts and protein levels of bronchoalveolar lavage fluid, but significantly increased total glutathione and taurine levels in bronchoalveolar lavage fluid.
These results indicate that oral N-acetylcysteine improves the pulmonary antioxidant protection and may be useful in reducing lung damage produced by bleomycin.
Idiopathic pulmonary fibrosis is a chronic inflammatory interstitial lung disease of potential fatal prognosis and poor response to available medical therapy 1. It has been hypothesized that activated inflammatory cells which accumulate in the lower airways release increased amounts of reactive oxygen species (ROS) which, combined with a deficiency in glutathione, the major component of the lung antioxidant defense system, produces lung injury and fibrosis 2. The antioxidant N-acetylcysteine (NAC) has shown beneficial effects in diseases in which ROS appear involved 3. In short-term studies, NAC improved the antioxidant screen of the lung by elevating glutathione levels in patients with pulmonary fibrosis accompanied by better pulmonary function and low incidence of adverse effects 4–6.
One of the clinically important causative agents in pulmonary fibrosis is bleomycin, an antineoplastic drug widely used in experimental models of pulmonary fibrosis resembling human disease 7, and used to assess potential therapeutic agents including NAC 8–14. NAC exerts direct antioxidant properties as a free radical scavenger and, as an l-cysteine prodrug, increases reduced glutathione in airway cells under oxidant stress 3, 15. In addition, benefit could be afforded by increased synthesis of taurine following NAC administration 16, since taurine protects against oxidant damage in the lung 17.
The aim of the present study was to examine the effects of orally administered NAC in a rat model of lung injury produced by endotracheal bleomycin. Lung damage was assessed by a semiquantitative histological score, and lung hydroxyproline was measured as a marker of collagen deposition. Cell counts, protein and glutathione levels in bronchoalveolar lavage fluid (BALF) were also determined. In addition, the changes in taurine levels in BALF, plasma and granulocytes were measured following NAC administration.
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Materials and methods |
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Animal model
Pathogen-free, male Sprague-Dawley rats, weighing 225–250 g at the start of experiments, were obtained from B&K Universal G.J.S.L. (Barcelona, Spain). Rats were fed rodent chow (A04; Panlab, Barcelona, Spain) and were maintained in a 12-h light-dark cycle. This study conformed to European Community (Directive 86/609/EEC) and Spanish guidelines for the use of experimental animals and it was approved by the institutional committee of animal care and research.
To produce pulmonary fibrosis, animals received endotracheally, by the transoral route, a single sublethal dose of bleomycin (2.5 U·kg–1 dissolved in 0.25 mL of 0.9% NaCl). Control animals were subjected to the same protocol but received the same volume of intratracheal saline instead of bleomycin. Tracheal instillation was carried out under halothane anaesthesia. Rats were weighed every 3 days. Fourteen days after endotracheal bleomycin or saline, the animals were killed by a lethal injection of sodium pentobarbital followed by exsanguination from abdominal aorta. Bronchoalveolar lavage was performed and lungs were weighed and processed separately for biochemical and histological studies as indicated below. The 14th day after bleomycin was selected as the approximate time for maximal rate of collagen synthesis 13.
Experimental groups
The animals were randomly divided into four groups (group A: vehicle+vehicle; group B: NAC+vehicle; group C: vehicle+bleomycin; and group D: NAC+bleomycin). Treatments (vehicle or NAC) were administered orally by gavage on a daily basis (at 9:00 h) from 7 days prior to the intratracheal instillation of bleomycin or saline up to the conclusion of the experiments 14 days postinstillation. Oral NAC was selected as the usual way of administration in the clinical setting 4, 6, and also because this route had not been previously examined in this rat model. The oral dose of NAC was 3 mmol·kg–1 per day, given as a single dose in a final volume of 1 mL of distilled water as vehicle. The dose level and schedule were based on previous studies 11–13.
Additional experiments were carried out to examine the influence of dose level of NAC and its time of administration as follows: 1) a lower dose of oral NAC (0.3 mmol·kg–1) was administered daily from 7 days prebleomycin to 14 days post-bleomycin; and 2) oral NAC (3 mmol·kg–1·day–1) was given from one day before or 7 days after bleomycin challenge to 14 days postbleomycin. Control groups received vehicle+vehicle and vehicle+bleomcyin. In these additional experiments, hydroxyproline quantification was used to assess pulmonary fibrosis.
Bronchoalveolar lavage
BALF was obtained by washing the right lung four times with 4 mL aliquots of saline through a tracheal cannula. Cell suspensions were concentrated by low speed centrifugation, and the cell pellet resuspended. Total cell counts were made in a haemocytometer. Differential cell counts were determined from cytospin preparations by counting 300 cells stained with May-Grünwald-Giemsa. Total protein content in BALF supernantants was measured by adding 10 µL of each sample to 90 µL of 0.9% NaCl together with 1 mL of Coomassie blue reagent (Bio-Rad; Munich, Germany). Absorbances were determined at 595 nm using a spectrophotometer.
Histological assessment
For histological studies, the left lung was first perfused by its main bronchus with a fixative solution (10% neutral-buffered formalin) at a pressure of 25 cmH2O, immersed in the fixative for 12–24 h, and blocks taken. Tissue blocks were placed in formalin, dehydrated in a graded series of ethanols, embedded in paraffin, cut into 4 µm-thick serial sections, and stained with haematoxylin-eosin and Masson's trichrome to identify inflammatory cells, connective tissue and collagen deposition. Histologic grading of lesions was performed by two experienced histopathologists using a blinded semiquantitative scoring system for extent and severity of inflammation and fibrosis in lung parenchyma, as previously outlined 18. Briefly, three lung sections from each animal were systematically scanned using a x10 objective and each successive field was scored using the following grading scheme: grade 0 for normal tissue, grades 1–4 for presence of pulmonary inflammation and fibrosis. The severity of lesions was graded as 1 (mild), 2 (moderate), 3 (severe) and 4 (severe inflammation accompanied by total distortion of structure). The extent of lesions was graded as 1 (<10% of the slide), 2 (10–40%), 3 (40–70%), and 4 (>70% of tissue affected). Fields predominantly occupied by portions of large bronchi or vessels were not counted. The pattern of distribution of the lesions was defined as multifocal and/or diffused, with or without affectation of the subpleural zone. Oedema was scored progressively as perivascular (grade 1), interstitial (grade 2), intra-alveolar (grade 3) and organized oedema (grade 4). Infiltration of inflammatory cells was graded 0–4 relating to their increasing presence in the interstitial, peribronchiolar and intra-alveolar spaces by counting each cell type in 10 random fields.
Biochemical studies
Lung hydroxyproline content was measured as outlined by Woessner 19. Samples were homogenized and then hydrolyzed in 6 N HCl for 18 h at 110°C. The hydrolysate was then neutralized with 2.5 M NaOH. Aliquots (2 mL) were analysed for hydroxyproline content after the addition of 1 mL of chloramine T, 1 mL of perchloric acid, and 1 mL of dimethylaminobenzladehyde. Samples were read for absorbance at 550 nm in a spectrophotometer. Results are expressed as mg of hydroxyproline per lung.
Total glutathione was measured in aliquots of BALF using the glutathione reductase-5,5'-dithiobis-(2-nitrobenzoic acid) recycling assay described by Tietze 20, and the results are expressed as glutathione equivalents (2GSH+GSSG) in nmol·mL–1.
Taurine levels were measured in BALF, blood plasma and peripheral polymorphonuclear leukocytes. These cells were selected as relevant to the bleomycin model 14, and influenced by taurine 21. Taurine was measured by a fluorometric technique that uses a derivatization with o-phthaldialdehyde prior to high-performance liquid chromatography (HPLC) 22. The limit of detection of this technique has been established at 5 pmol per analysis which is adequate for levels found in BALF, plasma and granulocytes of rodents 17, 21.
Statistical analysis of results
Data are expressed as mean±sem. Statistical analysis was carried out by analysis of variance (ANOVA) followed by appropriate post hoc tests including Bonferroni correction and unpaired t-test. Significance was accepted when p<0.05.
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). The rats in group C (vehicle+bleomycin) failed to gain weight during the first week after bleomycin instillation; thereafter weight gain paralleled that observed in rats not exposed to bleomycin. A similar, but less marked trend was noticed for rats in group D (NAC+bleomycin; fig. 1).
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). In rats exposed to bleomycin and treated with NAC, there was a tendency for lower lung weights but statistical significance was not reached. The lung hydroxyproline levels, a marker of collagen deposition, were increased approximately two-fold in bleomycin-treated rats compared to rats that received saline intratracheally. Treatment with NAC significantly reduced the hydroxyproline content, although it remained higher than levels in rats not exposed to bleomycin (fig. 2b).
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. The total cell count was significantly increased in bleomycin-treated rats compared to in rats not exposed to bleomycin. The differential cell count showed that neutrophils were markedly augmented while the changes in the rest of the cell types failed to reach significance. NAC was without effect on total and differential cell numbers (table 1).
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Histopathology
Masson trichrome and haematoxylin-eosin stained lung sections were examined by light microscopy to determine whether bleomycin-induced pulmonary fibrosis was decreased by treatment with NAC. Lungs from rats in groups A (vehicle+vehicle) and B (NAC+vehicle) were histologically normal (not shown). Lungs from rats in group C (vehicle+bleomycin) showed marked peribronchiolar and interstitial infiltration with inflammatory cells (predominantly mononuclear cells including macrophages and lymphocytes with fewer numbers of neutrophils and scattered eosinophils), extensive cellular thickening of interalveolar septa, interstitial oedema, increases in interstitial cells with a fibroblastic appearance and in interstitial collagen deposition detected by the trichrome stain, and association with focal cuboidal metaplasia of alveolar lining cells (fig. 3a and 3b). The pattern of distribution of lesions was multifocal (i.e. patchy areas of pulmonary fibrosis) in most cases, commonly involving the pleura.
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). A semiquantitative score of the severiry and extent of inflammation and fibrosis showed that most of the indices were reduced in group D compared to group C, yet significance was reached only for macrophages and the severity and extension of fibrosis (table 2).
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shows that taurine levels were increased in groups treated with NAC but not exposed to bleomycin. Bleomycin alone also increased taurine levels in BALF, and NAC treatment produced a further increase of BALF taurine levels.
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). By contrast, the taurine levels measured in granulocytes did not show significant changes in rats irrespective of bleomycin exposure or treatment with NAC (fig. 5c).
Influence of dose level and time of administration of N-acetylcysteine
In these experiments, the lung hydroxyproline content in untreated rats exposed to bleomycin was 4,229±203 µg·lung–1 (n=6, p<0.05 compared to a value of 2,298±154 µg·lung–1 obtained in control rats not challenged with bleomycin, n=6). Rats receiving a lower dose of NAC (0.3 mmol·kg–1) from 7 days prebleomycin, showed no reduction in lung hydroxyproline (4,090±189 µg·lung–1; n=6, p>0.05 compared to vehicle+bleomycin). When NAC (3 mmol·kg–1) was given from one day prebleomycin, hydroxyproline values were decreased (3,232±110 µg·lung–1; n=6, p<0.05 compared to vehicle+bleomycin) but the same dose of NAC administered from 7 days postbleomycin instillation failed to reduce hydroxyproline levels (4,301±244 µg·lung–1; n=6, p>0.05 compared to vehicle+bleomycin).
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Discussion |
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The potential of NAC to attenuate lung damage and subsequent fibrosis remains controversial in the literature. Thus, in the rat, intratracheal NAC (
200 mg·kg–1) abolished the lung injury subsequent to simultaneously administered bleomycin (5 U·kg–1, intratracheal) 8 but intraperitoneal or subcutaneous NAC (200 mg·kg–1·day–1) started before bleomycin (5 U·kg–1, intratracheal), failed to ameliorate lung toxicity 9, 10. In hamsters, NAC (200 mg·kg–1·day–1, intraperitoneal) did not reduce lung damage elicited by endotracheal bleomycin (7.5 U·kg–1) 12. In mice, oral NAC (400 mg·kg–1·day–1, before and after intratracheal bleomycin 7 U·kg–1) reduced lung collagen content, although lung wet weights and histopathology was not improved. 13. Also in mice, NAC (400 mg·kg–1) given as a single intraperitoneal dose prior to combined hyperbaric oxygen (445 kPa) and bleomycin (7 U·kg–1, endotracheal) was effective to protect against the lung damage that ensues these noxious stimuli 11. Recently, aerosolized NAC was reported to attenuate lung fibrosis produced by systemic bleomycin in mice 14. There are several possible explanations for these differing results. The observed effects will depend on the dose level of bleomycin, the timing between bleomycin and NAC administration, the animal species and strain, and even the criteria for detection of lung injury. The dose level of NAC shown to be effective in this study (490 mg·kg–1) is close to effective doses in mice 11, 13. A lower dose (50 mg·kg–1) was also found to be ineffective, in keeping with other studies 23. At the effective dose level, this beneficial effect of NAC was only partial, which is consistent with other recent reports 13, 14. The effects of higher doses of NAC were not tested, but beneficial effects appear insurmountable in other studies 11. This work used a dose of bleomycin (2.5 U·kg–1) lower than that used by others 8–13. This dose level causes no mortality in spite of producing pulmonary fibrosis consistently, and eases detection of pharmacological effects. Similar and lower intratracheal doses of bleomycin have recently been used for studies in this model 24.
The mechanism by which NAC limits fibrosis is unclear, but is likely to be via its ability to reduce damage to lung structures in the early stage of disease, since NAC administration from 7 days postbleomycin in the present study failed to influence the ensuing pulmonary fibrosis. This finding is consistent with a recent report showing that aerosolized NAC loses its effectiveness against pulmonary fibrosis when given after bleomycin administration 14. Pulmonary injury produced by bleomycin probably involves generation of oxidant species by an iron-dependent mechanism 7. Further damage is probably elicited by increased amounts of ROS, produced by activated inflammatory cells which accumulate in the pulmonary lesions induced by bleomycin 2. NAC may act directly as an oxygen radical scavenger but also, since it is a cell-permeable sulphydryl compound, readily enters cells and promotes the production of glutathione by furnishing its limiting precursor, l-cysteine 3. Both mechanisms may provide protection against bleomycin-predicted and leukocyte-mediated cytotoxicity in the lung.
The glutathione status has been studied in patients with idiopathic pulmonary fibrosis by Meyer and coworkers 4, 5 who found a deficiency in total glutathione levels in epithelial lining fluid but not in BALF. Oral treatment with NAC increased the total glutathione levels in the epithelial lining and BALF of patients with idiopathic pulmonary fibrosis, accompanied by an improvement of pulmonary function tests 4, 6. However, the pathogenic involvement of glutathione deficiency in lung fibrosis induced by bleomycin has scarcely been studied. Intratracheal bleomycin (5–7.5 U·kg–1) did not alter lung nonprotein sulphydryl in hamsters but intraperitoneal NAC (200–400 mg·kg–1·day–1 for 13 days) increased lung nonprotein sulphydryl 12. A tendency was found to lower glutathione levels in BALF of bleomycin exposed rats, as was a significant increase of glutathione in NAC-treated rats. These results suggest that replenishment of lung glutathione may have a role in the beneficial effect produced by NAC against the pulmonary toxicity elicited by bleomycin. However, further studies measuring the reduced and oxidized forms of gluthathione in BALF and lung tissue are required to ascertain the influence of glutathione redox balance in this animal model and in the beneficial effects of NAC.
In addition to replenishment of lung glutathione by NAC, this study also aimed to obtain an indication of the cysteine catabolism in stress conditions as produced by bleomycin administration. Taurine is the last metabolite of cysteine still maintaining the carbon chain 16, and posseses antioxidant properties and other regulatory functions in host defense 25. Administration of taurine has been found protective in vivo in bleomycin-, amiodarone-, and ozone-induced lung injury as well as against oxidant-induced lung epithelial damage in vitro 17, 25. Conversely, taurine levels in BALF are increased after bleomycin or ozone exposure 17, 25, in BALF of asthmatics 26, and in cystic fibrosis sputum 27. The precise mechanisms of the protective effects of taurine and the explanation for its increased levels in the inflammed lung are still unclear. The beneficial effects of taurine against bleomycin-induced pulmonary damage are ascribed to downregulation of overexpression of the procollagen gene and fibrogenic cytokines, and inhibition of nuclear factor-kappa B activation, effects in which its antioxidant properties appear involved 28.
The present study has confirmed and extended the observation of increased taurine levels in BALF and plasma to bleomycin-exposed rats. NAC further enhanced taurine levels in BALF and plasma of rats receiving bleomycin. Therefore, this amino acid may contribute to the beneficial effect of NAC in this rat bleomycin model. The mechanism underlying this additional augmentation of taurine in the NAC-treated rat is uncertain, but alteration of cysteine catabolism with increased taurine formation under inflammatory conditions has been recently reported 29.
N-acetylcysteine has also been reported to interfere with a number of inflammatory cytokines involved in lung fibrogenesis 14, 30 and to block the in vivo activation of nuclear factor-kappa B in rat lungs 23. Further studies are warranted to elucidate the mechanism of the beneficial effect of N-acetylcysteine in this model, as well as the potential for N-acetylcysteine administration as an adjunt therapy for patients with fibrosing alveolitis 6, including that produced during bleomycin treatment.
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B activation and neutrophilic lung inflammation. J Immunol 1996;157:1630–1637.[Abstract]
expression in bleomycin-induced lung injury. J Leukoc Biol 1998;64:528–536.[Abstract]
B in mice. J Pharmacol Exp Ther 2000;293:82–90.This article has been cited by other articles:
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 E. Barreiro, D. Sanchez, J. B. Galdiz, S. N. A. Hussain, J. Gea, and on behalf of the ENIGMA in COPD project N-acetylcysteine increases manganese superoxide dismutase activity in septic rat diaphragms Eur. Respir. J., December 1, 2005; 26(6): 1032 - 1039. [Abstract] [Full Text] [PDF]
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S. Blesa, J. Cortijo, M. Mata, A. Serrano, D. Closa, F. Santangelo, J.M. Estrela, J. Suchankova, and E.J. Morcillo Oral N-acetylcysteine attenuates the rat pulmonary inflammatory response to antigen Eur. Respir. J., March 1, 2003; 21(3): 394 - 400. [Abstract] [Full Text] [PDF]
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A Serrano-Mollar, D Closa, J Cortijo, E J Morcillo, N Prats, M Gironella, J Panes, J Rosello-Catafau, and O Bulbena P-selectin upregulation in bleomycin induced lung injury in rats: effect of N-acetyl-L-cysteine Thorax, July 1, 2002; 57(7): 629 - 634. [Abstract] [Full Text] [PDF]
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: Group A (vehicle+vehicle); 
: Group B (acetylcysteine (NAC)+vehicle); •: Group C (vehicle+bleomycin); 
: Group D: NAC+bleomycin; 
: bleomycin or vehicle administration. b) Comparison of the gain in body weight at day 21 from day 6 indicating that rats exposed to bleomycin experienced less weight gain compared to unexposed rats. Although rats treated with NAC and exposed to bleomycin showed a tendency for greater weight gain, the difference failed to reach significance. All data are presented as mean±sem of 10 (A), 6 (B) and 11 (C and D) animals. *: p<0.05 versus A.
