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Am J Physiol Lung Cell Mol Physiol 292: L1095-L1104, 2007. First published January 12, 2007; doi:10.1152/ajplung.00283.2006
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Protective effect of orally administered carnosine on bleomycin-induced lung injury

Salvatore Cuzzocrea ,2,3,* Tiziana Genovese,2,3,* Marco Failla,1 Graziella Vecchio,4 Mary Fruciano,1 Emanuela Mazzon,2,3 Rosanna Di Paola,2,3 Carmelo Muià,2,3 Cristina La Rosa,1 Nunzio Crimi,1 Enrico Rizzarelli,4,5 and Carlo Vancheri1

1Department of Internal Medicine and Specialistic Medicine, Section of Respiratory Diseases, University of Catania, Catania, Italy; 2Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy; 3Istituto di Ricovero e Cura a Carattere Scientifico Centro Neurolesi "Bonino-Pulejo," Messina, Italy; 4Department of Chemical Sciences, University of Catania, Italy, Catania, Italy; 5Institute of Biostructures and Bioimages, Consiglio Nazionale delle Ricerche, Catania, Italy

Submitted 27 July 2006 ; accepted in final form 3 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), IL-1, and TNF-{alpha} in different experimental settings. In the present study, we investigated the efficacy of carnosine on the animal model of bleomycin-induced lung injury. Mice were subjected to intratracheal administration of bleomycin and were assigned to receive carnosine daily by an oral bolus of 150 mg/kg. One week after fibrosis induction, bronchoalveolar lavage (BAL) cell counts and TGF-beta levels, lung histology, and immunohistochemical analyses for myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase were performed. Finally, apoptosis was quantified by terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay. After bleomycin administration, carnosine-treated mice exhibited a reduced degree of lung damage and inflammation compared with wild-type mice, as shown by the reduction of 1) body weight, 2) mortality rate, 3) lung infiltration by neutrophils (myeloperoxidase activity and BAL total and differential cell counts), 4) lung edema, 5) histological evidence of lung injury and collagen deposition, 6) lung myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase immunostaining, 7) BAL TGF-beta levels, and 8) apoptosis. Our results indicate that orally administered carnosine is able to prevent bleomycin-induced lung injury likely through its direct antioxidant properties. Carnosine is already available for human use. It might prove useful as an add-on therapy for the treatment of fibrotic disorders of the lung where oxidative stress plays a role, such as for idiopathic pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.

lung fibrosis; oxidative stress; chelating agent

IDIOPATHIC PULMONARY FIBROSIS (IPF) is the most common among interstitial pneumonias of unknown origin and one of the most aggressive interstitial lung diseases. It is characterized by a chronic and progressive course leading to respiratory failure with a median survival under 3 yr (1, 43). Pathological findings in this disease include temporally and spatially nonhomogeneous areas of inflammation, fibrosis, and honeycombing. Microscopically, the hyperplasia of type II pneumocytes and active fibroblasts proliferation leads to excessive matrix deposition resulting in the irreversible distortion of the lung architecture (56), which in turn is responsible for impaired gas exchanges and respiratory failure.

A number of exogenously administered agents, including commonly used drugs, are known to induce a iatrogenic form of pulmonary fibrosis (57). Bleomycin is an efficacious antitumor agent currently used in humans. Nevertheless, repeated administration of this drug may lead to lung inflammation and fibrosis as a side effect. Because this phenomenon is easily reproduced in different mammals, intratracheal administration of bleomycin has become the most widely used experimental model of lung fibrosis, although with certain limitations. This model is characterized by an early neutrophilic response, increased collagen deposition, and fibroblast proliferation (13). Bleomycin alters the balance between oxidants and antioxidant defense systems in the lung. In this particular organ, the selective absence of bleomycin hydrolase activity gives a high susceptibility to bleomycin-induced oxidative stress (20). Contemporarily, hydroxyl radicals, superoxide anion radical, hydrogen peroxide, and peroxynitrite are increased by bleomycin administration (51). Reactive oxygen species (ROS) overproduction ultimately results in tissue injury, with activation of several intracellular signaling pathways leading to the production of proinflammatory cytokines (33). DNA is a target for ROS activity as well. Radical oxygen species production, by determination of DNA damage, in turn activates poly(ADP-ribose) polymerase (PARP). This largely expressed nuclear protein contributes to the maintenance of genomic stability and to the repair of oxidative DNA damage (5). Although PARP activity promotes cell survival, PARP activation depletes NAD+ and decreases ATP levels, thus leading to cell death after extensive DNA strand breaks (14). Therefore, ROS produced in response to oxidative stress are able to contribute by multiple pathways to the pathogenesis of bleomycin-induced lung injury.

Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine, which is present abundantly in muscle and nervous tissue in many species (52). It acts as a physiological buffer, a metal ion chelator, a free radical scavenger, and finally as an antioxidant (17, 35). Besides the known anti-aging properties of this dipeptide, it has been demonstrated that carnosine plays a role in inflammation. In fact, carnosine-inhibited hydrogen peroxide induced IL-8 release in vitro (58). IL-6 and TNF-{alpha} were reduced by the oral administration of carnosine in an animal model of diabetes (44), and finally carnosine proved to decrease the secretion of transforming growth factor-beta (TGF-beta) and of various extracellular matrix components induced by high doses of glucose in vitro (34).

Given the antioxidant and anti-inflammatory properties of carnosine, we sought to investigate carnosine efficacy on lung injury caused by bleomycin administration. To this end, we evaluated the following endpoints: 1) loss of body weight, 2) mortality rate, 3) infiltration of the lung with polymorphonuclear neutrophils (myeloperoxidase activity), 4) edema formation, 5) histological evidence of lung injury, 6) bronchoalveolar lavage (BAL) inflammatory cells counts, 7) TGF-beta expression, inducible nitric oxide synthase (iNOS) activity, nitrotyrosine formation, and activation of the nuclear enzyme PARP, and 8) lung cells apoptosis [terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL) assay].


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male CD mice (25–35 g; Harlan Nossan) were housed in a controlled environment and provided with standard rodent chow and water. The University of Messina Review Board (Italy) approved the study for the care of animals. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the European Economic Community (EEC) regulations (Official Journal of EECL 358/1 12/18/1986).

Experimental groups. Mice were randomly allocated into the following groups. 1) Wild-type (WT) + bleomycin group: mice were subjected to bleomycin-induced lung injury (n = 15). 2) WT + saline group: this was a sham-operated group in which identical surgical procedures to the bleomycin group was performed, except that saline was administered instead of bleomycin (n = 15). 3) Carnosine group: mice in this group were treated the same as the WT + bleomycin group; however, mice were administered daily with an oral bolus of carnosine in PBS to a final dose of 150 mg/kg (Sigma, Milan, Italy) starting 30 min after bleomycin intratracheal administration. 4) Vehicle group: mice in this group were treated the same as carnosine group mice, except that only PBS was administered as control vehicle instead of carnosine (n = 15).

Induction of lung injury by bleomycin. Mice received a single intratracheal instillation of saline (0.9%) or saline containing bleomycin sulfate (1 mg/kg body wt) in a volume of 50 µl and were killed after 7 days by pentobarbital sodium overdose.

Measurement of fluid content in lung. The wet lung weight was measured after careful excision of extraneous tissues. The lung was exposed for 48 h at 180°C, and the dry weight was measured. Water content was calculated by subtracting dry weight from wet weight.

Histological examination. Lung biopsies were taken 7 days after injection of bleomycin. Lung biopsies were fixed for 1 wk in 10% (wt/vol) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). After specimens were embedded in paraffin, the sections were prepared and stained by trichrome stain. All sections were studied by light microscopy (Dialux 22 Leitz). The severity of fibrosis was semiquantitatively assessed according to the method proposed by Ashcroft et al. (2).

Briefly, the grade of lung fibrosis was scored on a scale from 0 to 8 by examining randomly chosen sections, with fields per sample at a magnification of x100. Criteria for grading lung fibrosis were as follows: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; and grade 8, total fibrous obliteration of the fields.

Immunohistochemical localization of iNOS, nitrotyrosine, PARP, and TGF-beta. Tyrosine nitration, an index of the nitrosylation of proteins by peroxynitrite and/or ROS, was determined by immunohistochemistry as previously described (15). At the end of the experiment, the tissues were fixed in 10% (wt/vol) PBS-buffered formaldehyde, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% hydrogen peroxide (vol/vol) in 60% (vol/vol) methanol for 30 min. The sections were permeabilized with 0.1% (wt/vol) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with anti-iNOS rat polyclonal antibody (1:500 in PBS, vol/vol), anti-nitrotyrosine rabbit polyclonal antibody (1:500 in PBS, vol/vol) or anti-poly(ADP-ribose) goat polyclonal antibody (1:500 in PBS, vol/vol), and finally anti-TGF-beta rabbit polyclonal antibody (1:500 in PBS, vol/vol). Sections were washed with PBS and incubated with appropriate secondary antibodies. Specific labeling was detected with biotin-conjugated IgG and avidin-biotin peroxidase complex (DBA). To confirm that the immunoreaction for the nitrotyrosine was specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PARP, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out.

TUNEL assay. TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instructions (Apotag, horseradish peroxidase kit; DBA). Briefly, sections were incubated with 15 µg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% hydrogen peroxide for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase buffer containing deoxynucleotidyl transferase and biotinylated dUTP in terminal deoxynucleotidyltransferase buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.

Myeloperoxidase activity assay. Myeloperoxidase activity, an indicator of polymorphonuclear leukocyte accumulation, was determined as previously described (49). At the specified time after injection of bleomycin, lung tissues were obtained and weighed, and each piece was homogenized in a solution containing 0.5% (wt/vol) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol of peroxide per minute at 37°C and was expressed in milliunits per gram of wet tissue.

BAL. Seven days after bleomycin or saline solution instillation, mice were euthanized, and the trachea was immediately cannulated with an intravenous polyethylene catheter (Neo Delta Ven 2; delta Med, Viadana, Italy) equipped with a 24-gauge needle on a 1-ml syringe. Lungs were lavaged once with 0.5 ml D-PBS (GIBCO, Paisley, UK). In >95% of the mice, the recovery volume was over 0.4 ml. The BAL fluid was spun at 800 rpm, the supernatant was removed, and the pelleted cells were collected. Total BAL cells were enumerated by counting on a hemocytometer in the presence of trypan blue. Cytospins were prepared from resuspended BAL cells.

Cytospins of BAL cells were made by centrifuging 50,000 cells onto microscope slides using Shandon Cytospin 3 (Shandon, Astmoore, UK). Slides were allowed to air dry and were then stained with Diff-Quick stain set (Baxter Scientific, Miami, FL). A total of 400 cells were counted from randomly chosen high-power microscope fields for each sample.

BAL supernatants were collected and analyzed in duplicate by ELISA for biologically active TGF-beta1 quantification (TGF-beta1 Emax immunoassay system, Promega Italia, Milan, Italy), in accordance with manufacturer's instructions.

Materials. Unless otherwise stated, all compounds were obtained from Sigma-Aldrich (Poole, Dorset, UK). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Scientific).

Statistical evaluation. All values are expressed as means ± SE of n observations. For the in vivo studies, n represents the total number of animals studied; dead animals were replaced in further experiments to reach the specified number of observations. In the experiments involving histology or immunohistochemistry, results shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A P value of <0.05 was considered significant. Statistical analysis for survival data was calculated by Fisher's exact probability test. For such analyses, P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Histological examination of lung from bleomycin-treated animals revealed significant tissue damage (Fig. 1A). Compared with lung sections taken from saline-treated animals, these were characterized by extensive inflammatory infiltration of neutrophils, lymphocyte and plasma cells extending through the lung epithelium, and fibrosis and granulomas visible in the perivascular region. The administration of the dipeptide carnosine in mice significantly prevented lung inflammation induced by bleomycin administration. This was confirmed by the histological grading of lung fibrosis according to criteria of Ashcroft et al. (2) executed on Masson's trichrome-stained slides, showing a significant reduction of the fibrosis score in carnosine-treated animals (Fig. 1B).


Figure 1
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  		Fig. 1. Effect of carnosine on lung fibrosis. A: bleomycin administration in wild-type (WT) mice caused extensive inflammation, inflammatory cells infiltration, and fibrosis. Carnosine-treated animals showed patchy areas of inflammation with minimal fibrosis. Each image (trichrome stain) is representative of at least 3 experiments performed on different experimental days. Original magnification = x150. B: histological grading of lung fibrosis. Black bar, vehicle group; gray bar, carnosine group. Data are means ± SE from 15 mice for each group. *P < 0.01 vs. sham. °P < 0.01 vs. bleomycin.

 
Bleomycin in WT mice elicited an inflammatory response characterized by the accumulation of water in lung as an indicator of tissue edema and neutrophil infiltration in lung tissues as demonstrated by the myeloperoxidase assay (Fig. 2). Carnosine significantly reduced the fluid content and the neutrophil infiltration in bleomycin-treated animals.


Figure 2
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  		Fig. 2. Effect of carnosine on tissue edema and myeloperoxidase activity in the lung. Bleomycin in WT mice elicited an inflammatory response characterized by the accumulation of water in lung (A) and increased myeloperoxidase activity (B). Carnosine significantly reduced the edema formation and myeloperoxidase activity. Black bar, vehicle group; gray bar, carnosine group. Data are means ± SE from 15 mice for each group. *P < 0.01 vs. sham. °P < 0.01 vs. bleomycin.

 
Accordingly, with the extensive infiltration of inflammatory cells, bleomycin instillation in WT mice produced a significant increase of inflammatory cells compared with sham WT mice (10.2 ± 0.2 vs. 1.69 ± 0.3 cells x 105/ml; P < 0.001) (Fig. 3A). Carnosine-treated WT mice that underwent bleomycin intratracheal instillation showed a reduced number of inflammatory cells in BAL compared with bleomycin WT mouse group (5.15 ± 0.6 vs. 10.2 ± 0.2 cells x 105/ml; P < 0.05). Differential cell counts showed a similar profile compared with all of the sham groups. In bleomycin-treated WT mice vs. sham WT mice, respectively, it was evident that there were increased numbers of macrophages (5.77 ± 0.6 vs. 2.06 ± 0.3 cells x 105/ml; P < 0.001), lymphocytes (1.49 ± 0.4 vs. 0.18 ± 0.1 cells x 105/ml; P < 0.01), and neutrophils (3.66 ± 1.1 vs. 0.10 ± 0.04 cells x 105/ml; P < 0.01).


Figure 3
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  		Fig. 3. Effect of carnosine on bleomycin-induced total and differential cellularity in bronchoalveolar lavage (BAL). A: total BAL cellularity for sham and bleomycin-treated mice with and without carnosine administration. B: differential cells counts for macrophages, lymphocytes, neutrophils, and eosinophils per ml of BAL fluid. Open bar, sham group; black bar, bleomycin group; gray bar, carnosine group. Data, expressed as means ± SE, are representative of 15 mice for each group. +P < 0.001 vs. sham; ++P < 0.05 vs. bleomycin-treated WT; °P < 0.01 vs. sham; *P < 0.05 vs. bleomycin-treated WT.

 
Carnosine-treated mice that underwent bleomycin tracheal instillation showed a decreased content of BAL inflammatory cells when evaluated by differential cell count on cytospin preparations (Fig. 3B). In these mice, macrophages (4.09 ± 0.6 vs. 5.77 ± 0.6 cells x 105/ml; P < 0.05), lymphocytes (0.52 ± 0.1 vs. 1.49 ± 0.3 cells x 105/ml; P < 0.05), and neutrophils (0.77 ± 0.3 vs. 3.66 ± 1.1 cells x 105/ml; P < 0.05) were significantly reduced compared with bleomycin-treated WT group. Eosinophils did not show any statistically significant difference among all groups.

Immunohistochemical analysis of lung sections obtained from bleomycin-treated mice revealed a positive staining for iNOS in macrophages and neutrophils present in the alveolar space and in septal walls (Fig. 4A). Carnosine treatment abolished immunostaining for iNOS in lungs of animals treated with bleomycin. We then assessed the nitration of protein by nitrotyrosine immunohistochemical staining on tyrosine residues. Lung sections obtained from bleomycin-treated mice revealed a positive staining for nitrotyrosine mostly in the inflammatory cell infiltrate present in the interstitium and also in the alveolar pneumocyte layer (Fig. 4B). In contrast, no staining for nitrotyrosine was found in lungs of bleomycin-treated mice that underwent carnosine treatment. Moreover, mice treated with bleomycin exhibited a substantial increase in lung PARP staining (Fig. 4C), mainly present in inflammatory cells of the interstitium and in the alveolar pneumocyte layer at the nuclear level. Carnosine administration abolished the increased staining for PARP in lung section of bleomycin-treated mice. Finally, we studied total TGF-beta1 in lung sections by immunohistochemistry and active TGF-beta1 in BAL supernatants by ELISA. Bleomycin induced a remarkable increase of TGF-beta1 staining in the alveolar epithelium and in the inflammatory infiltrate.


Figure 4
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  		Fig. 4. Effect of carnosine on lung inducible nitric oxide synthase (iNOS), nitrotyrosine, and poly(ADP-ribose) polymerase (PARP) immunostaining. A: after bleomycin injection in WT mice, a positive staining for iNOS in macrophages and neutrophils was present in the alveolar space and in septal walls. Carnosine treatment abolished immunostaining for iNOS in the lung of animals treated with bleomycin. B: bleomycin administration increased nitrotyrosine staining mainly in inflammatory infiltrate present in the interstitium but also in the alveolar pneumocyte layer. In contrast, no staining for nitrotyrosine was found in the lungs of bleomycin-treated mice that underwent carnosine treatment. C: mice treated with bleomycin exhibited a substantial increase in lung PARP staining in inflammatory cells of the interstitium but also in the alveolar pneumocyte layer at the nuclear level. Carnosine administration abolished the increased staining for PARP in lung sections of bleomycin-treated mice. Original magnification = x150. These microphotographs are representative of at least 3 experiments performed on different experimental days.

 
In contrast, carnosine-treated mice did not exhibit such an increase (Fig. 5A). Similarly, biologically active TGF-beta1 levels of the bleomycin-operated animals were significantly reduced in the BAL of the carnosine group (652 ± 38 vs. 158 ± 68 pg/ml; P < 0.01; Fig. 6). There was no iNOS, nitrotyrosine, PARP, or TGF-beta1 increase in the lungs of sham-operated animals (Figs. 4 and 5). In WT mice, the severe lung injury caused by bleomycin administration was associated with a significant increase of apoptosis, as demonstrated by the marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments (Fig. 5B). In contrast, no apoptotic cells or fragments were observed in lungs obtained from carnosine-treated mice. Similarly, no apoptotic cells were observed in lungs from sham animals.


Figure 5
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  		Fig. 5. Effect of carnosine on lung transforming growth factor-beta1 (TGF-beta1) immunostaining and terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL) assay. A: after bleomycin injection in WT mice, a positive staining for TGF-beta1 in the inflammatory infiltrate and in the alveolar epithelium was present in lung sections. Carnosine treatment strongly diminished immunostaining for TGF-beta1 in the lung of animal treated with bleomycin. B: bleomycin-induced apoptosis was measured by TUNEL-like staining. One week after bleomycin administration, lung tissue demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments. No apoptotic cells or fragments were observed in tissues obtained from bleomycin mice treated with carnosine. Similarly, no apoptotic cells were observed in sections obtained from sham animals. Original magnification = x150. These microphotographs are representative of at least 3 experiments performed on different experimental days.

 

Figure 6
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  		Fig. 6. BAL active TGF-beta levels. Bleomycin in WT mice elicited a relevant increase TGF-beta levels in BAL. Carnosine significantly reduced biologically active TGF-beta levels after 1 wk from bleomycin administration. Black bar, vehicle group; gray bar, carnosine group. Data are means ± SE from 15 mice for each group. *P < 0.01 vs. sham. °P < 0.01 vs. bleomycin.

 
In WT mice, the severe lung injury caused by bleomycin administration was associated with a significant loss in body weight and increased mortality (Fig. 7). Carnosine administration in bleomycin-treated mice significantly attenuated the loss in body weight and abolished bleomycin-induced mortality at 7 days.


Figure 7
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  		Fig. 7. Effect of carnosine on body weight and mortality rate. A: body weight was recorded immediately before bleomycin administration and daily during the experimental period. Carnosine administration significantly prevented the loss of body weight. B: survival was significantly improved in carnosine-treated mice compared with mortality rate of the bleomycin-treated WT mice. {blacklozenge}, Bleomycin group; bullet, carnosine-treated animals. Data are means ± SE from 15 mice for each group. *P < 0.01 vs. vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Lung fibrosis is the common endpoint of an heterogeneous group of pathological entities termed interstitial lung diseases, which are characterized by chronic inflammation and progressive fibrosis of the pulmonary interstitium (28). Although the fibrotic process has greatly attracted research efforts, there is nowadays strong evidence that the cellular redox state and the oxidant/antioxidant balance play important roles in the pathogenesis of IPF. Indeed, oxidative stress induces apoptosis of structural cells and upregulates the synthesis of proinflammatory cytokines (45). In BAL fluid and in plasma from patients with IPF, an imbalance between oxidant and antioxidants has been proven (53). Myeloperoxidase, which is a marker of neutrophil activity, is strongly increased in the BAL of these patients (29). Similarly, iNOS, which is the main source of NO during inflammation, and nitrotyrosine, a by-product of peroxynitrite activity, are upregulated in IPF lungs (41, 55).

Intratracheal instillation of the antitumour agent bleomycin is the most commonly used animal model for pulmonary fibrosis. Earlier reports point out that the pathogenesis of bleomycin-induced fibrosis, at least in part, is mediated through the generation of ROS, which cause the peroxidation of membrane lipids and DNA damage (26).

Carnosine is a dipeptide (beta-alanyl-L-histidine) discovered nearly 100 years ago. Since then, many functions have been proposed for this compound, including wound healing promoter, ion-chelant agent, antioxidant, and free-radical scavenger (52). Carnosine prevents cellular toxicity in vitro, with a direct anti-peroxidative activity on proteins (31), lipids (50), and DNA bases (38). The antioxidant and metal ion-chelator properties of carnosine have been successfully tested on animal models of stomatitis and duodenal and gastric ulcers and on different ocular disorders (3, 36). Furthermore, carnosine has been proven to affect inflammation directly by modulating cytokine release. In an animal model of diabetes, carnosine reduced IL-6 and TNF-{alpha} (44), whereas TGF-beta and extracellular matrix deposition were reduced by carnosine after stimulation with high doses of glucose in vitro (34).

In the present study, we show a significant reduction of tissue damage and cellular apoptosis in lungs of bleomycin-treated mice treated with carnosine. Not only the extracellular matrix deposition evaluated histologically in lung sections of treated mice showed a reduced degree of fibrosis but also the alveolar architecture was preserved, indicating that the treatment with this antioxidant significantly prevented lung damage induced by bleomycin.

Furthermore, carnosine proved efficacious to significantly lower total and biologically active TGF-beta1 levels. TGF-beta1 plays a central role in fibrotic disorders in different organs, including fibrosis of the lung. In fact, it stimulates collagen and fibronectin production in fibroblasts (21); on the other hand, it can suppress the production of proteases that degrade the extracellular matrix (59). TGF-beta1 has been shown to be increased in bleomycin-induced lung fibrosis in the alveolar inflammatory infiltrate (32). Secretion of active TGF-beta1 by alveolar macrophages is augmented after bleomycin administration in mice, whereas latent TGF-beta1 secretion remains elevated for a prolonged length of time, and it is probable that the extent of inflammation and fibrosis in this model depend on the quantity of active TGF-beta1 available (37). Finally, the increase of TGF-beta1 mRNA precedes the biosynthesis of type I and type III procollagen in lung fibrosis (32).

Lung edema and fall of body weight were virtually absent, and inflammation was significantly reduced in carnosine-treated animals. Leukocytes recruited into the tissue can contribute to tissue destruction by the production of reactive oxygen metabolites, granule enzymes, and cytokines that further amplify the inflammatory response. These responses are an integral part of the antibacterial defenses. For example, neutrophil-derived myeloperoxidase uses hydrogen peroxide produced by dismutation of superoxide to produce hypochlorous acid, a compound with relevant antibacterial properties (62). On the other hand, ROS can mediate tissue injury. An increased susceptibility to bleomycin has been reported in mice lacking extracellular superoxide dismutase (SOD1), which indicates that superoxide anion radicals play a main role in experimental fibrosis (19). Recently, carnosine and some carnosine derivatives have been shown to scavenge superoxide anion radicals (39) and to chelate copper(II), leading to a complex that shows SOD1-like activity with a catalytic constant equal to that found for native SOD1 (6, 7). Moreover, this natural dipeptide has been shown to protect primary astroglial cell cultures from iNOS-induced oxidative stress by scavenging nitric oxide radicals (12). Superoxide reacts with nitric oxide to generate highly reactive metabolites such as peroxynitrite. This compound is able to oxidize proteins, resulting in direct nitration of tyrosine residues. Protein structure and function can be subsequently altered and enzymatic activity affected. Proteins containing nitrotyrosine residues have been detected in different pathologies associated with enhanced oxidative stress and increased levels of peroxynitrite (30). Consistent with these data, carnosine reduced the expression of iNOS and the nitration of tyrosine residues in lung sections. Nitric oxide mediates vaso- and bronchodilatation, and it is synthesized from L-arginine by two constitutive forms of NOS, which are involved in the physiological regulation of airway function (48). However, iNOS generates much larger quantities of nitric oxide than the constitutive isoforms, and it is directly involved in host defense from infections (46) and in various models of inflammation (16, 61). Exogenous nitric oxide is able to stimulate in vitro fibroblast proliferation (24), whereas iNOS upregulation in lung fibroblasts is associated with the early proliferative response to cytokine stimulation (54). Finally, the pharmacological inhibition and the genetic disruption of iNOS have been shown to reduce the development of inflammatory responses and fibrosis in lung of bleomycin-treated animals (25).

Nitrotyrosine immunostaining was initially proposed as a relatively specific marker for the detection of the endogenous formation of peroxynitrite (4). There is, however, recent evidence that certain other reactions can also induce tyrosine nitration; e.g., the reaction of nitrite with hypochlorous acid and the reaction of myeloperoxidase with hydrogen peroxide can lead to the formation of nitrotyrosine (18). Increased nitrotyrosine staining is considered, therefore, as an indication of "increased nitrative stress" rather than a specific marker of the generation of peroxynitrite. Nevertheless, our results confirm previous data on the activity of carnosine on tyrosine nitration in vitro (22).

We finally tested a novel pathway of inflammation that relies on the nuclear PARP enzyme activation by superoxide and peroxynitrite. PARP contributes to the maintenance of genomic stability and to the repair of oxidative DNA damage (5). However, its activity can deplete NAD+ and interfere with glycolysis and ATP metabolism, ultimately leading to cell death because of extensive DNA strand breaks (14).

Although the exact role of PARP in human lung fibrosis has not been investigated, it has been shown that PARP is implicated in experimental fibrosis and that PARP inhibition confers protection from inflammation and fibrosis in different animal models, including the bleomycin model (60). Indeed, we have previously shown that PARP is elevated in the lung of bleomycin-treated mice (25). In this study, we do confirm that bleomycin administration increased PARP levels, whereas carnosine greatly reduced the expression of this enzyme. Interestingly, treated animals showed a reduced degree of lung cellular apoptosis. Therefore, it is conceivable that the protection from bleomycin injury might derive, at least in part, from the carnosine scavenging ability on superoxide and peroxynitrite, which in turn prevents PARP activation and the depletion of crucial metabolites to cellular activities.

The beneficial activity of carnosine administration after bleomycin treatment was reflected by some favorable clinical outcomes. Most notably, the beneficial effects given by this treatment resulted in the complete abrogation of the bleomycin-induced mortality.

Together, our data further support the rationale for antioxidant therapy in interstitial lung diseases.

Similarly to other proposed therapeutic chelating molecules, carnosine and its derivatives form very stable mono- and polynuclear copper complexes (40, 47), suggesting a dual action as an antioxidant and also as a chelating agent. For example, tetrathiomolybdate is a copper-chelating agent (11) that has been recently proposed for the treatment of cancer (8) and against bleomycin-induced pulmonary fibrosis in mice (10). The tetrathiomolybdate metal-chelating affect lowers systemic copper(II) levels, thus inhibiting several proangiogenic cytokines in cancer (27) and modulating profibrotic and proinflammatory cytokines such as TGF-beta and TNF-{alpha} (9, 23, 42). Tetrathiomolybdate efficacy in experimental lung fibrosis has indeed been associated with a significant decrease of systemic copper level. Considering these results, carnosine may be considered a potential multifunctional drug with both chelating and antioxidant activity; these properties may prove useful for the treatment and the prevention of diseases in which ROS are thought to play a major role such as the interstitial pathologies of the lung.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was funded by Italian Ministry of University and Research (MIUR) Grants PRIN 2005, 2005069290_003, and FIRB RBNE03PX83_001.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Vancheri, Dept. of Internal Medicine and Specialistic Medicine, Respiratory Diseases Section, Univ. of Catania, Via Passo Gravina, 187, 95125 Catania, Italy (e-mail: vancheri{at}unict.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. Cuzzocrea and T. Genovese contributed equally to this work. Back


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