Melatonin limits lung injury in bleomycin treated mice

Introduction

Pulmonary fibrosis is a common response to various insultsto the lung and it is the end-point of a numerous andheterogeneous group of disorders known as interstitial lung

diseases (ILD) that are characterized by chronic inflamma-tion and progressive fibrosis of the pulmonary interstitium;the pathological changes occur in the alveolar walls (inclu-ding epithelial cells, and capillaries), septae, and the perivas-

cular, perilymphatic, and peribronchiolar connective tissues[1]. While the pathogenesis is incompletely understood, agrowing body of evidence suggests two different pathogenic

routes for developing pulmonary fibrosis. The inflammatorypathway, where a shift to the so-called T-helper 2 typecytokine network is critical, and the epithelial pathway

represented by idiopathic pulmonary fibrosis, by far themostaggressive ILD. Both routes may trigger a number ofcytokines/growth factors inducing fibroblast migration/

proliferation and phenotype change to myofibroblasts, witha consequent accumulation of extracellular matrix [2].

The common pathologic features in ILD include thefibrosis of the interstitium, involvement collagen, elastic

and smooth muscle elements, architectural remodeling ofthe interstitium, chronic inflammation of the interstitium(i.e. variable increases in lymphocytes, plasma cells, macr-

ophages, eosinophils, and mast cells), hyperplasia of type IIcells and hyperplasia of endothelial cells [1].

Intratracheal instillation of the antitumor agent bleomy-

cin (BLM) is the most commonly used animal model forpulmonary fibrosis [3]. BLM is a drug widely used as anantineoplastic agent in the treatment in the germ-line

tumors and Hodgkins lymphoma. An estimated 3–5% ofpatients develop pulmonary fibrosis causes a dose-depend-ent interstitial pulmonary fibrosis [4] and it can also cause

significant morbidity and mortality [5]. This model ischaracterized by an early predominantly neutrophilicinflammatory response, increased fibroblast proliferation,

and enhanced collagen deposition because of increasedcollagen synthesis and decreased collagen degradation [6].In addition, it has been shown that fibroproliferativeactivity coexists with inflammation, and that the major

proliferative phase occurs during the first week after BLMinduced injury [7].Inflammation is a major component in the pathogenesis

of interstitial lung disease that is orchestrated in part byendogenous and migrating leukocytes. These leukocytestogether with lung epithelial and endothelial cells produce a

feedback circle where stimuli from injury responses canactivate alveolar and interstitial macrophages. Activatedleukocytes release additional reactive oxygen and nitrogen

species and proteases that sustain the injury/repair proces-ses that are considered to contribute to the fibroticprocesses [8]. There are several lines of evidence thatimplicate superoxide (O��

2 ) as one the ROS (reactive oxygen

species) that mediate lung injury [9, 10]. The superoxideanion participates in the generation of other toxic metab-olites, most importantly hydrogen peroxide (H2O2), hydro-

xyl radical (�HO), and peroxynitrite (ONOO)).Some important pro-inflammatory roles for ROS include

endothelial cell damage and increased microvascular per-

meability [11], formation of chemotactic factors such asleukotriene B4 [12, 13], recruitment of neutrophils at sites of

Abstract: Melatonin is the principal secretory product of the pineal gland

and its role as an immuno-modulator is well established. Recent evidence

shows that melatonin is a scavenger of oxyradicals and peroxynitrite and

exerts protective effects in septic shock, hemorrhagic shock and

inflammation. The aim of this study was to investigate the effect of melatonin

on the lung injury caused by bleomycin (BLM) administration. Mice

subjected to intratracheal administration of BLM developed significant lung

injury characterized by a marked neutrophil infiltration [assessed by

myeloperoxidase (MPO) activity] and by tissue edema. In addition, an

increase of immunoreactivity to nitrotyrosine, poly-ADP-ribose (PAR) was

also observed in the lung of BLM-treated mice. Also, lung injury induced by

BLM administration was correlated with a significant loss of body weight

and with a significant mortality. Administration of melatonin (10 mg/kg i.p.)

daily significantly reduced the (i) loss of body weight, (ii) mortality rate, (iii)

infiltration of the lung with polymorphonuclear neutrophils (MPO activity),

(iv) edema formation and (v) histological evidence of lung injury.

Administration of melatonin also markedly reduced the nitrotyrosine and

PAR formation. Taken together, our results demonstrate that treatment with

melatonin significantly reduces lung injury induced by BLM in the mice.

Tiziana Genovese, Rosanna DiPaola, Emanuela Mazzon,Carmelo Muia, Achille P. Caputiand Salvatore Cuzzocrea

Department Clinical and Experimental

Medicine and Pharmacology, School of

Medicine, University of Messina Torre

Biologica, Policlinico Universitario, Messina,

Italy

Key words: bleomycin, interstitial lung

diseases, melatonin, oxidative stress, poly

(ADP-ribose) polymerase,

polymorphonuclear leukocyte

Address reprint requests to Salvatore Cuzzo-

crea, Department of Clinical and Experimental

Medicine and Pharmacology, School of Medi-

cine, University of Messina, Torre Biologica,

Policlinico Universitario Via C. Valeria, Gazzi,

98100 Messina, Italy.

E-mail: salvator@unime.it

Received November 30, 2004;

accepted January 26, 2005.

J. Pineal Res. 2005; 39:105–112Doi:10.1111/j.1600-079X.2005.00229.x

Copyright � Blackwell Munksgaard, 2005

Journal of Pineal Research

105

inflammation [14, 15], lipid peroxidation (LPO) and oxida-tion, DNA single-strand damage [16] and formation ofONOO), a potent cytotoxic and proinflammatory mole-

cules [15, 17]. The ROS and peroxynitrite also cause DNAdamage [18], which results in the activation of the nuclearenzyme poly (ADP-ribose) polymerase (PARP), depletionof NAD+ and adenosine triphosphate (ATP) and ulti-

mately cell death [18]. Thus, ROS are produced by multiplemechanisms in all cell compartments and play a significantrole in the pathogenesis of lung diseases where oxidant

stress is increased.In the last decade, melatonin has been found to be highly

protective against damage to macromolecules resulting

from oxygen and nitrogen-based reactants, as a direct freeradical scavenger [19, 20], as an indirect antioxidant whenstimulating antioxidant enzymes [20]. In fact, melatonin notonly scavenges oxygen free radicals such as O��

2 , �OH,

LOO�, and ONOO) [21], but can also enhance theantioxidative potential of the cell by stimulating thesynthesis of antioxidative enzymes like super oxide dismu-

tase, glutathione peroxidase (GPX), and also the enzymesthat are involved in the synthesis of glutathione. In manyinstances, melatonin increases the expression of mRNA and

the activities of the antioxidative enzymes [22].The purpose of this study was to determine whether

melatonin would ameliorate development of pulmonary

fibrosis in the mice. We evaluated the following endpointsof the inflammatory process: (a) loss of body weight, (b)mortality rate, (c) infiltration of the lung with polymor-phonuclear neutrophils (myeloperoxidase activity), (d)

edema formation, (e) histological evidence of lung injury,(f) tissue levels of tumor necrosis factor (TNF-a), and (g)interleukin-1 (IL-1b) expression, (h) nitrotyrosine forma-

tion and activation of the nuclear enzyme PARP.

Materials and methods

Materials

Unless otherwise stated, all compounds were obtained from

Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Mela-tonin was obtained from Alexis, Milan, Italy. All otherchemicals were of the highest commercial grade available.

All stock solutions were prepared in nonpyrogenic saline(0.9% NaCl; Baxter, Italy, UK).

Animals

Male CD mice (25–35 g; Harlan Nossan, Milan, Italy) were

housed in a controlled environment and provided withstandard rodent chow and water. Animal care was incompliance with Italian regulations on protection ofanimals used for experimental and other scientific purpose

(D.M. 116192) as well as with the EEC regulations (O.J. ofE.C. L 358/1 12/18/1986).

Experimental groups

Mice were randomly allocated into the following groups:

(i) BLM + vehicle group. Mice were subjected to BLM-induced lung injury and received 10% ethanol in saline, i.p.

bolus 30 min after the instillation of BLM and every 24 hrstarting from day 1 until 14th day (n ¼ 30), (ii) melatoningroup. Same as the BLEO Bleomycin + vehicle group but

were administered melatonin (10 mg/kg) i.p. bolus 30 minafter the instillation of BLM and every 24 hr starting fromday 1 until 14th day (n ¼ 30), (iii) Sham + vehicle group.Sham-operated group in which identical procedures to the

BLM group was performed, except that the saline wasinstilled instead of BLM and received the vehicle formelatonin i.p. bolus 30 min after the instillation of saline

and every 24 hr starting from day 1 until 14th day, (iv)Sham + melatonin group. Identical to Sham + vehiclegroup, but were administered melatonin i.p. bolus every

24 hr starting from day 1 until 14th day (n ¼ 30). Thevarious groups of mice were observed for 15 days in orderto determine survival differences. The dose of melatoninwas choosing in agreement with our pleurisy study [23].

Induction of lung injury by BLM

Mice received by a catheter (tracheotomy tube), a singleintratracheal instillation of saline (0.9%) or saline contain-ing BLM sulfate (0.1 U/mouse) at end-expiration in a

volume of 100 lL, and the liquid was followed immediatelyby 300 lL of air to ensure delivery to the distal airways andwere killed after 15 days by pentobarbitone overdose.

Measurement of water content in lung

The wet lung weight was measured after careful excision of

extraneous tissues. The lung was washed at 180�C for 48 hrand the dry weight was measured. Water content wascalculated by subtracting dry weight from wet weight.

Histological examination

Lung biopsies were taken 15 days after injection of BLM.Lung biopsies were fixed for 1 wk in 10% (w/v) PBS-buffered formaldehyde solution at room temperature,dehydrated using graded ethanol and embedded in Para-

plast (Sherwood Medical, Mahwah, NJ, USA). Sectionswere then deparaffinized with xylene, stained with vanGieson’s solution. All sections were studied using light

microscopy (Dialux 22 Leitz Ziess, Milan, Italy).

Immunohistochemical localization of nitrotyrosineand PARP

Tyrosine nitration, an index of the nitrosylation of proteins

by ONOO) and/or ROS, was determined by immunohist-ochemistry as previously described [24]. At the end of theexperiment, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 8 lm sections were prepared

from paraffin embedded tissues. After deparaffinization,endogenous peroxidase was quenched with 0.3% (v/v)H2O2 in 60% (v/v) methanol for 30 min. The sections were

permeablized with 0.1% (w/v) Triton X-100 in PBS for20 min. Nonspecific adsorption was minimized by incuba-ting the section in 2% (v/v) normal goat serum in PBS for

20 min. Endogenous biotin or avidin binding sites wereblocked by sequential incubation for 15 min with biotin

Genovese et al.

106

and avidin (DBA, Milan, Italy), respectively. Sections wereincubated overnight with anti-nitrotyrosine rabbit poly-clonal antibody (1:500 in PBS, v/v) or with anti-poly (ADP-

ribose) goat polyclonal antibody rat (1:500 in PBS, v/v).Sections were washed with PBS, and incubated withsecondary antibody. Specific labelling was detected with abiotin-conjugated goat anti-rabbit IgG and avidin–biotin

peroxidase complex (DBA). In order to confirm that theimmunoreaction for the nitrotyrosine was specific somesections were also incubated with the primary antibody

(anti-nitrotyrosine) in the presence of excess nitrotyrosine(10 mm) to verify the binding specificity. To verify thebinding specificity for PAR some sections were also

incubated with only the primary antibody (no secondary)or with only the secondary antibody (no primary). In thesesituations no positive staining was found in the sectionsindicating that the immunoreaction was positive in all the

experiments carried out.

Myeloperoxidase activity

The MPO activity, an indicator of polymorphonuclearleukocyte (PMN) accumulation, was determined as pre-

viously described [25]. At the specified time followinginjection of BLM, lung tissues were obtained andweighed; each piece homogenized in a solution containing

0.5% (w/v) hexadecyltrimethyl-ammonium bromide dis-solved in 10 mm potassium phosphate buffer (pH 7) andcentrifuged for 30 min at 20,000 g at 4�C. An aliquot ofthe supernatant was then allowed to react with a solution

of tetramethylbenzidine (1.6 mm) and 0.1 mm H2O2. Therate of change in absorbance was measured spectropho-tometrically at 650 nm. The MPO activity was defined as

the quantity of enzyme degrading 1 lmol of peroxide/minat 37�C and was expressed in milliunits per gram of wettissue.

Measurement of cytokines

The levels of TNF-a and IL-1b was measured in the lung

tissue collected at the end of the study (at the day 15) byusing a colorimetric commercial solid phase ELISA kit

(Celbio Euroclone, Milan, Italy). The intra- and intercoef-ficient of variations were within 10%. The limit ofsensitivity of the assay was 10 pg/mL.

Statistical evaluation

All values in the figures and text are expressed as

mean ± S.E.M. of �n� observations. For the in vivo studies,�n� represents the number of animals studied. In theexperiments involving histology or immunohistochemistry,

the figures shown are representative of at least threeexperiments 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. Statisticalanalysis for survival data was calculated by Fisher’s exactprobability test. The Mann–Whitney test was used to

examine differences between the body weight and organweights of control and experimental groups.

Results

Histological examination of lung sections revealed signifi-

cant tissue damage (Fig. 1A). Thus, when compared withlung sections taken from sham-treated animals (data notshown), histological examination of lung sections of mice

treated with BLM showed edema, tissue injury as well asinfiltration of the tissue with PMN (Fig. 1A). Administra-tion of melatonin significantly prevented lung damageinduced by BLM administration (Fig. 1B). Furthermore,

the injection of BLM elicited an inflammatory responsecharacterized by the accumulation of water in lung as anindicator of edema (Fig. 2). The treatment with melatonin

significantly reduced the edema formation.Infiltration of leukocytes into the lung tissue is believed

to contribute significantly to the tissue injury and dysfunc-

tion associated with pulmonary fibrosis, as activated PMNrelease large amounts of free radicals. The increase in tissuelevels of MPO activity at day 15 after the instillation ofBLM, indicated an important neutrophil infiltration

(Fig. 3). Treatment of BLM-treated mice with melatoninsignificantly reduced the neutrophil infiltration (Fig. 3).

Fig. 1. Effect of melatonin on lung injury and histological score. Lung injury was produced after bleomycin administration characterized byinflammatory cells infiltration, fibrosis as well as an extensive areas of collagen (A). The treatment with melatonin (B) significantly reducedthe extent and severity of the histological signs of lung injury. Figure is representative of at least three experiments performed on differentexperimental days.

Effect of melatonin on lung injury

107

A significant increase of TNF-a and IL-1b levels wereobserved in the lung tissue from all mice at day 15 after theinstillation of BLM (Fig. 4). In contrast, the levels of these

pro-inflammatory cytokines were significantly lower in themelatonin-treated mice (Fig. 4). No significant cytokinesincrease was observed in the lung tissue of sham-treated

mice.Immunohistochemical analysis of lung sections obtained

from mice treated with BLM also revealed a positivestaining for nitrotyrosine (Fig. 5A) mainly localized in

pneumocyte membrane as well as in inflammatory cells (seearrows). In contrast, no positive staining for nitrotyrosinewas found in the lungs of BLM-treated mice, which had

been treated with melatonin (Fig. 5B). Immunohistochem-ical analysis of lung sections obtained from mice treatedwith BLM also revealed a positive staining for PAR

(Fig. 5C) mainly localized in the nuclei of the pneumocyteas well as in inflammatory cells localized in perivasculararea (see arrows). In contrast, no staining for PAR wasfound in the lungs of BLM-treated mice treated with

melatonin (Fig. 5D). There was no staining for eithernitrotyrosine or PAR in lungs obtained from the shamgroup of mice (data not shown).

In vehicle-treated mice, the severe lung injury caused byBLM administration was associated with a significant lossin body weight (Fig. 6A). The treatment with melatonin

significantly reduced the loss in body weight (Fig. 6A). TheBLM-treated mice, which had received vehicle, developedsevere lung injury and 40% of these animals died within

15 days, respectively, after BLM administration (Fig. 6B).In contrast, none of the mice, which had been treated withmelatonin died (Fig. 6B).

Discussion

This study examined the beneficial effect of melatonin on

BLM-induced pulmonary fibrosis and, in particular ourresults indicate that melatonin has strong anti-inflamma-tory properties resulting in a reduced: (a) nitrotyrosine and

PAR formation, (b) infiltration of the lung with polymor-phonuclear neutrophils (MPO activity), (c) tissue levels ofTNF-a and IL-1b, (d) edema formation, (e) tissue injury, (f)loss of body weight, and (g) mortality rate.

The BLM–oxygen complex is thought to bind to DNAand lead to the efficient cleavage of the phosphodiester–deoxyribose backbone and the generation of ROS [26]. In

the presence of O2 and a reducing agent, the ferrous ion–BLM complex becomes activated and functions mechanic-ally as a ferrous oxidase, transferring electrons from ferrous

ion to O2 to produce ROS that cause scission of DNA

Fig. 2. The injection of bleomycin elicited an inflammatoryresponse characterized by the accumulation of water in lung as anindicator of edema. The treatment with melatonin significantlyreduced the edema formation. Data are mean values ± S.E. meanvalues from nine mice for each group. *P < 0.01 versus sham;�P < 0.01 versus bleomycin.

Fig. 3. Effect of melatonin on myeloperoxidase (MPO) activity inthe lung. The MPO actvity in the lungs of bleomycin (BLM)-treated mice were significantly increased in comparison with sham-operated mice. Melatonin treatment significantly reduced theBLM-induced increase in MPO activity. Data are mean val-ues ± S.E. mean values from nine mice for each group. *P < 0.01versus sham; �P < 0.01 versus BLM.

Fig. 4. Lung tumor necrosis factor(TNF)-a (A) and interleukin (IL)1-b (B)production at 15 days after bleomycin(BLM) administration. Cytokine levelswere significantly reduced in melatonin-treated mice. Data are mean val-ues ± S.E.M. of nine mice for eachgroup. *P < 0.01 versus sham; �P < 0.01versus BLM.

Genovese et al.

108

[27, 28]. Therefore, earlier reports [29, 30] point out that thepathogenesis of BLM-induced fibrosis, at least in part, ismediated through the generation of ROS which cause theperoxidation of membrane lipids and DNA damage. If this

is valid, then antioxidant therapy may prevent the lungfibrosis caused by BLM and may prevent other diseasesrelated with interstitial pulmonary fibrosis. Because BLM

administration results in increased LPO and alters activitiesof antioxidant enzymes in bronchoalveolar lavage fluidsand lung tissue [31, 32], in previous studies [33, 34], some

natural or synthetic antioxidants have been used to protectagainst BLM oxidative lung toxicity both in vivo and alsoin vitro. Numerous studies have clearly demonstrated theprotective effects of melatonin in various conditions of

oxidant stress, including H2O2 induced lipid peroxidation[35]. More recently, it also has been shown that melatoninacts as a modestly potent ONOO) scavenger, and protects

cultured cells against ONOO)-induced injury [36]. Amechanism by which melatonin attenuates inflammationis by reducing ROS and ONOO) formation [37]. This is

Fig. 5. Immunohistochemical localization of nitrotyrosine and poly-ADP-ribose (PAR) in the lung. After bleomycin (BLM) injection,positive staining for nitrotyrosine (A) and for PAR (C) was localized mainly in nuclei of inflammatory cells. There was a marked reductionin the immunostaining for nitrotyrosine (B) and for PAR (D) in the lungs of BLM-treated mice treated with melatonin. This figure isrepresentative of at least three experiments performed on different experimental days.

Fig. 6. Body weight (A) was recorded immediately before bleomycin (BLM) administration and daily for all the experimental period.Melatonin treatment significantly prevent the loss of body weight (A). Survival is significantly improved in melatonin-treated mice incomparison with the high mortality rate of the BLM-treated mice (B). Data are mean values ± S.E. mean values from 30 (original number)mice for each group. *P < 0.01 versus BLM.

Effect of melatonin on lung injury

109

important as the pro-inflammatory and cytotoxic effects ofROS and ONOO) are numerous [38]. Removal of ONOO)

by agents such as FeTMPS, a porphyrin-containing mole-

cule which increases the rate of isomerization of ONOO) tonitrate is cytoprotective and anti-inflammatory [39, 40].The ONOO) also nitrosates tyrosine residues in proteins

and nitrotyrosine formation along with its detection by

immunofluorescence has been used as a marker for thedetection of the endogenous formation of ONOO) [41].Using nitrotyrosine as a marker for the presence of ONOO)

has been challenged by the demonstration that otherreactions can also induce tyrosine nitration, e.g. thereaction of nitrite with hypochlorous acid and the reaction

of MPO with H2O2 leads to the formation of nitrotyrosine[42]. Thus, increased nitrotyrosine staining is considered asan indicator of �increased nitrosative stress� rather than aspecific marker of the generation of ONOO) [42]. We have

found that nitrotyrosine is indeed present in lung sectionstaken after BLM administration and that melatoninreduced the staining in these tissues.

This effect is likely related to a direct scavenging effect ofmelatonin on ONOO), as demonstrated in our in vitrostudies [36]. Additional protective effects of melatonin may

lie within the ability of this indole to reduce oxyradical-related oxidant processes by either directly interfering withthe oxidants [21, 43, 44], or up-regulating antioxidant

systems such as superoxide dismutase [22, 45, 46] orenhancing the catalytic activity of GPX [47]. In addition,recently Arslan et al. [28] have clearly demonstrated thatmelatonin administration prior to BLM reversed glutathi-

one GSH depletion, and attenuated the increases in theactivities of antioxidant defense enzymes, except that ofGSH-Px.

A novel pathway of inflammation, governed by thenuclear enzyme PARP has been proposed in relation to�OH- and ONOO)-induced DNA single strand breakage

[48]. Continuous or excessive activation of PARP, producesextended chains of ADP-ribose on nuclear proteins andresults in a substantial depletion of intracellular NAD+

and subsequently, ATP, leading to cellular dysfunction andultimately cell death [49]. This pathway plays an importantrole in various forms of inflammation [18, 23]. It isconceivable that by scavenging �OH and ONOO), melato-

nin would prevent the DNA single strand breakage [36],and thus prevent the activation of PARP in inflammation.We also demonstrate here that melatonin reduced the

increase in PARP activation in the lung from BLM-treatedmice. Thus, we propose that the anti-inflammatory effectsof melatonin may be at least in part because of the

prevention of the activation of PARP.Besides attenuating ONOO) production and PARS

activation, melatonin also reduced the development ofedema, neutrophil accumulation and had an overall

protective effect on the degree of lung injury as assessedby histological examination. The recruitment of inflamma-tory cells like neutrophils, is responsible for production of

several immunomodulatory factors, such as cytokines andlipid mediators. Among the cytokines, TNF-a and IL-1 areparadigmatic pro-inflammatory mediators responsible for

leukocyte activation and recruitment [50, 51]. Therefore,there is good evidence that TNF-a and IL-1b, are clearly

involved in the pathogenesis of lung fibrosis as thesecytokines are present in lung tissues and can be detectedimmunohistochemically in the inflamed tissues [52]. Direct

evidence that TNF-a and IL-1b play a role in thepathogenesis of experimental lung injury has been obtainedin animal models in which blocking of the action of thesecytokines has been shown to delay the onset of experimen-

tal lung injury, suppress inflammation, and ameliorate lungdestruction that corresponds to the anti-inflammatoryresponse [53]. We confirm in the present study that the

model of lung injury used here leads to a substantialincrease in the levels of TNF-a and IL-1 in the lung afterBLM administration and we report that the production of

the pro-inflammatory cytokines are is significantly attenu-ated by the treatment with melatonin. Lissoni et al. [54]have clearly demonstrated that the administration ofmelatonin significantly reduced TNF-a levels in humans

with neoplasms.These data support the hypothesis that melatonin is an

inhibitor of BLM-induced lung fibrosis [28] and this

protective effect is observed also by a significant reductionof the edema formation, tissue damage and reduced contentof collagen like also other authors have demonstrated in the

same model of IPF damage [28]. Also the treatment withmelatonin reduced the loss of body weight and improvedthe survival of the mice.

Although the data support the view that melatonin haspotent anti-inflammatory effects, the importance of endog-enous melatonin as an anti-inflammatory factor remains tobe confirmed. Interesting data have recently been published

in this respect, describing that melatonin-deficient rats haveincreased lung injury after carrageenan administration [55].Based on the current results, we propose that the mode of

melatonin’s anti-inflammatory action is, at least in part,related to interference with ROS and ONOO)-relatedpathways.

References

1. Green FH. Overview of pulmonary fibrosis. Chest 2002;

122:334S–339S.

2. Pardo A, Selman M. Molecular mechanisms of pulmonary

fibrosis. Front Biosci 2002; 7:d1743–d1761.

3. Chandler DB, Hyde DM, Giri SN. Morphometric estimates

of infiltrative cellular changes during the development of

bleomycin-induced pulmonary fibrosis in hamsters. Am J

Pathol 1983; 112:170–177.

4. White DA, Schwartzberg LS, Kris MG et al. Acute chest

pain syndrome during bleomycin infusions. Cancer 1987;

59:1582–1585.

5. Crooke ST, Bradner WT. Bleomycin, a review. J Med 1976;

7:333–428.

6. Lazenby AJ, Crouch EC, Mcdonald JA et al. Remodeling

of the lung in bleomycin-induced pulmonary fibrosis in the rat.

An immunohistochemical study of laminin, type IV collagen,

and fibronectin. Am Rev Respir Dis 1990; 142:206–214.

7. Walsh J, Absher M, Kelley J. Variable expression of

platelet-derived growth factor family proteins in acute lung

injury. Am J Respir Cell Mol Biol 1993; 9:637–644.

8. Oury TD, Thakker K, Menache M et al. Attenuation of

bleomycin-induced pulmonary fibrosis by a catalytic anti-

Genovese et al.

110

oxidant metalloporphyrin. Am J Respir Crit Care Med 2001;

164:164–169.

9. Pron G, Belehradek J Jr, Orlowski S et al. Involvement of

membrane bleomycin-binding sites in bleomycin cytotoxicity.

Biochem Pharmacol 1994; 48:301–310.

10. Halliwell B, Gutteridge JMC. Free radicals. In: Biology

and Medicine. Oxford University Press, ed. Oxford University

Press, Oxford, UK, 1999. pp. 381–396.

11. Haglind E, Xia G, Rylander R. Effects of antioxidants and

PAF receptor antagonist in intestinal shock in the rat. Circ

Shock 1994; 42:83–91.

12. Fantone JC, Ward PA. A review: role of oxygen-derived free

radicals and metabolites in leukocyte-dependent inflammatory

reactions. Am J Pathol 1982; 107:395–418.

13. Li Y, Ferrante A, Poulos A et al. Neutrophil oxygen radical

generation. Synergistic responses to tumor necrosis factor and

mono/polyunsaturated fatty acids. J Clin Invest 1996; 97:1605–

1609.

14. Boughton-Smith NK, Evans SM, Laszlo F et al. The

induction of nitric oxide synthase and intestinal vascular per-

meability by endotoxin in the rat. Br J Pharmacol 1993;

110:1189–1195.

15. Salvemini D, Wang ZQ, Stern MK et al. Peroxynitrite

decomposition catalysts: therapeutics for peroxynitrite-medi-

ated pathology. Proc Natl Acad Sci USA 1998; 95:2659–2663.

16. Dix TA, Hess KM, Medina MA et al. Mechanism of site-

selective DNA nicking by the hydrodioxyl (perhydroxyl) rad-

ical. Biochemistry 1996; 35:4578–4583.

17. Beckman JS, Crow LP. Pathological implications of nitric

oxide, superoxide and peroxynitrite formation. Biochem Soc

Trans 1993; 21:330–334.

18. Cuzzocrea S, Caputi AP, Zingarelli B. Peroxynitrite-

mediated DNA strand breakage activates poly (ADP-ribose)

synthetase and causes cellular energy depletion in carrageenan-

induced pleurisy. Immunology 1998; 93:96–101.

19. Hardeland R, Reiter RJ, Poeggeler B et al. The signifi-

cance of the metabolism of the neurohormone melatonin:

antioxidative protection and formation of bioactive sub-

stances. Neurosci Biobehav Rev Fall 1993; 17:347–357.

20. Tan DX, Chen LD, Poeggler B et al. Melatonin: a potent,

endogenous hydroxyl radical scavenger. Endocrine J 1993;

1:57–60.

21. Allegra M, Reiter RJ, Tan DX et al. The chemistry of

melatonin’s interaction with reactive species. J Pineal Res 2003;

34:1–10.

22. Rodriguez C, Mayo JC, Sainz RM et al. Regulation of

antioxidant enzymes: a significant role for melatonin. J Pineal

Res 2004; 36:1–9.

23. Cuzzocrea S, Zingarelli B, Gilad E et al. Protective effect

of melatonin in carrageenan-induced models of local inflam-

mation: relationship to its inhibitory effect on nitric oxide

production and its peroxynitrite scavenging activity. J Pineal

Res 1997; 23:106–116.

24. Cuzzocrea S, Ianaro A, Wayman NS et al. The cyclopen-

tenone prostaglandin 15-deoxy-delta-(12,14)-PGJ2 attenuates

the development of colon injury caused by dinitrobenzene

sulphonic acid in the rat. Br J Pharmacol 2003; 138:678–688.

25. Mullane KM, Kraemer R, Smith B. Myeloperoxidase

activity as a quantitative assessment of neutrophil infiltration

into ischemic myocardium. J Pharmacol Methods 1985;

14:157–167.

26. Hecht SM. Bleomycin: new perspectives on the mechanism of

action. J Nat Prod 2000; 63:158–168.

27. Burger RM, Projan SJ, Horwitz SB et al. The DNA clea-

vage mechanism of iron-bleomycin. Kinetic resolution of

strand scission from base propenal release. J Biol Chem 1986;

261:15955–15959.

28. Arslan SO, Zerin M, Vural H et al. The effect of melatonin

on bleomycin-induced pulmonary fibrosis in rats. J Pineal Res

2002; 32:21–25.

29. Slosman DO, Costabella PM, Roth M et al. Bleomycin

primes monocytes-macrophages for superoxide production.

Eur Respir J 1990; 3:772–778.

30. Goodman MT, Hernandez B, Wilkens LR et al. Effects of

beta-carotene and alpha-tocopherol on bleomycin-induced

chromosomal damage. Cancer Epidemiol Biomarkers Prev

1998; 7:113–117.

31. Giri SN, Chen ZL, Younker WR et al. Effects of intratra-

cheal administration of bleomycin on GSH-shuttle enzymes,

catalase, lipid peroxidation, and collagen content in the lungs

of hamsters. Toxicol Appl Pharmacol 1983; 71:132–141.

32. Karam H, Hurbain-Kosmath I, Housset B. Antioxidant

activity in alveolar epithelial type 2 cells of rats during the

development of bleomycin injury. Cell Biol Toxicol 1998;

14:13–22.

33. Ikezaki S, Nishikawa A, Enami T et al. Inhibitory effects

of the dietary antioxidants butylated hydroxyanisole and

butylated hydroxytoluene on bronchioloalveolar cell proli-

feration during the bleomycin-induced pulmonary fibro-

sing process in hamsters. Food Chem Toxicol 1996; 34:

327–335.

34. Venkatesan N, Punithavathi V, Chandrakasan G.

Curcumin protects bleomycin-induced lung injury in rats. Life

Sci 1997; 61:PL51–PL58.

35. Sewerynek E, Abe M, Reiter RJ et al. Melatonin adminis-

tration prevents lipopolysaccharide-induced oxidative damage

in phenobarbital-treated animals. J Cell Biochem 1995;

58:436–444.

36. Gilad E, Cuzzocrea S, Zingarelli B et al. Melatonin is a

scavenger of peroxynitrite. Life Sci 1997; 60:PL169–PL174.

37. Cuzzocrea S, Reiter RJ. Pharmacological actions of mela-

tonin in acute and chronic inflammation. Curr Top Med Chem

2002; 2:153–165.

38. Squadrito GL, Pryor WA. The formation of peroxynitrite in

vivo from nitric oxide and superoxide. Chem Biol Interact

1995; 96:203–206.

39. Salvemini D, Jensen MP, Riley DP et al. Therapeutic

manipulations of peroxynitrite. Drug News Persp 1998;

11:204–214.

40. Misko TP, Highkin MK, Veenhuizen AW et al. Character-

ization of the cytoprotective action of peroxynitrite decom-

position catalysts. J Biol Chem 1998; 273:15646–15653.

41. Beckman JS. Oxidative damage and tyrosine nitration from

peroxynitrite. Chem Res Toxicol 1996; 9:836–844.

42. Eiserich JP, Hristova M, Cross CE et al. Formation of

nitric oxide-derived inflammatory oxidants by myeloperoxi-

dase in neutrophils. Nature 1998; 391:393–397.

43. Reiter RJ, Melchiorri D, Sewerynek E et al. A review of

the evidence supporting melatonin’s role as an antioxidant.

J Pineal Res 1995; 18:1–11.

44. Marshall KA, Reiter RJ, Poeggeler B et al. Evaluation of

the antioxidant activity of melatonin in vitro. Free Radic Biol

Med 1996; 21:307–315.

45. Barlow-Walden LR, Reiter RJ, Abe M et al. Melatonin

stimulates brain glutathione peroxidase activity. Neurochem

Int 1995; 26:497–502.

Effect of melatonin on lung injury

111

46. Antolin I, Rodriguez C, Sainz RM et al. Neurohormone

melatonin prevents cell damage: effect on gene expression for

antioxidant enzymes. FASEB J 1996; 10:882–890.

47. Pablos MI, Agapito MT, Gutierrez R et al. Melatonin

stimulates the activity of the detoxifying enzyme glutathione

peroxidase in several tissues of chicks. J Pineal Res 1995;

19:111–115.

48. Szabo C, Dawson VL. Role of poly(ADP-ribose) synthetase

in inflammation and ischaemia-reperfusion. Trends Pharmacol

Sci 1998; 19:287–298.

49. Chiarugi A. Poly(ADP-ribose) polymerase: killer or conspir-

ator? The �suicide hypothesis� revisited. Trends Pharmacol Sci

2002; 23:22–129.

50. Streiter RM. Inflammatory mechanisms are not a minor

component of the pathogenesis of idiopathic pulmonary

fibrosis. Am J Respir Crit Care Med 2002; 165:1206–1207.

51. Davies HR, Richeldi L, Walters EH. Immunomodulary

agents for idiopathic pulmonary fibrosis. Cochrane Database

Syst Rev 2003; 3:CD003134.

52. Pan LH, Ohtani H, Yamauchi K et al. Co-expression of

TNF alpha and IL-1 beta in human acute pulmonary fibrotic

diseases: an immunohistochemical analysis. Pathol Int 1996;

46:91–99.

53. Goldstein RH, Fine A. Potential therapeutic initiatives for

fibrogenic lung diseases. Chest 1995; 108:848–855.

54. Lissoni P, Pittalis S, Ardizzoia A et al. Prevention of cy-

tokine-induced hypotension in cancer patients by the pineal

hormone melatonin. Support Care Cancer 1996; 4:313–316.

55. Cuzzocrea S, Mazzon E, Dugo L et al. Protective effects of

n-acetylcysteine on lung injury and red blood cell modification

induced by carrageenan in the rat. FASEB J 2001; 15:1187–

1200.

Genovese et al.

112