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www.elsevier.com/locate/burns

Burns 31 (2005) 587–596

Leukotriene receptor blocker montelukast protects against

burn-induced oxidative injury of the skin and remote organs

Goksel Sener a,*, Levent Kabasakal a, Sule Cetinel b,Gazi Contuk b, Nursal Gedik c, Berrak C. Yegen d

a School of Pharmacy, Department of Pharmacology, Marmara University, 34668 Haydarpasa, Istanbul, Turkeyb School of Medicine, Departments of Histology and Embryology, Marmara University, 34668 Haydarpasa, Istanbul, Turkey

c School of Medicine, Department of Physiology, Marmara University, 34668 Haydarpasa, Istanbul, Turkeyd Kasımpasa Military Hospital, Division of Biochemistry, Istanbul, Turkey

Accepted 11 January 2005

Abstract

Thermal injury elicits several systemic consequences, among them the systemic inflammatory response where the generation of

reactive oxygen radicals and lipid peroxidation play important roles. In the present study, we investigated whether the leukotriene receptor

blocker montelukast is protective against burn-induced remote organ injury. Under brief ether anaesthesia, shaved dorsum of the rats was

exposed to 90 8C (burn group) or 25 8C (control group) water bath for 10 s. Montelukast (10 mg/kg) or saline was administered

intraperitoneally immediately after and at the 12th hour of the burn injury. Rats were decapitated 24 h after burn injury and the tissue

samples from lung, liver, kidney and skin were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels,

myeloperoxidase (MPO) activity and collagen contents. Tissues were also examined microscopically. Serum aspartate aminotransferase

(AST), alanine aminotransferase (ALT) levels and creatinine, urea (BUN) concentrations were determined to assess liver and kidney

function, respectively. Tumor necrosis factor-a (TNF-a) and lactate dehydrogenase (LDH) were also assayed in serum samples. Severe

skin scald injury (30% of total body surface area) caused a significant decrease in GSH level, which was accompanied with significant

increases in MDA level, MPO activity and collagen content of tissues. Similarly, serum ALT, AST and BUN levels, as well as LDH and

TNF-a, were elevated in the burn group as compared to control group. On the other hand, montelukast treatment reversed all these

biochemical indices, as well as histopathological alterations, which were induced by thermal trauma. Findings of the present study suggest

that montelukast possesses an anti-inflammatory effect on burn-induced damage in remote organs and protects against oxidative organ

damage by a neutrophil-dependent mechanism.

# 2005 Elsevier Ltd and ISBI. All rights reserved.

Keywords: Burn injury; Montelukast; Neutrophil; Oxidative damage; TNF-a; Glutathione

1. Introduction

Thermal trauma, one of the most common problems

faced in the emergency room, may cause damage to

multiple organs distant from the original burn wound and

may lead to multiorgan failure. Burn trauma produces

significant fluid shifts that, in turn, reduce cardiac output

and tissue perfusion and thus causes ischemia of the tissues

* Corresponding author. Tel.: +90 216 414 29 62; fax: +90 216 345 29 52.

E-mail addresses: gsener@marmara.edu.tr, gokselsener@hotmail.com

G. Sener).

305-4179/$30.00 # 2005 Elsevier Ltd and ISBI. All rights reserved.

oi:10.1016/j.burns.2005.01.012

[1]. While aggressive postburn volume replacement

increases oxygen delivery to previously ischemic tissue,

this restoration of oxygen delivery is thought to initiate a

series of deleterious events that exacerbate tissue injury

that occurred during low flow state [2].

The inflammatory response to burn is extremely

complex, resulting in local tissue damage and deleterious

systemic effects in all the organ systems distant from the

original wound. Several studies have demonstrated that

burn injury is associated with lipid peroxidation, which is an

autocatalytic mechanism leading to oxidative destruction of

cellular membranes, and their destruction can lead to the

G. Sener et al. / Burns 31 (2005) 587–596588

production of toxic, reactive metabolites and cell death

[3,4]. A growing body of evidence suggests that the

activation of a proinflammatory cascade after major burn is

responsible for the development of immune dysfunction,

and susceptibility to sepsis and multiple organ failure [1]. In

addition to the well-documented role of neutrophils and

endothelial cells in oxidative tissue damage or organ failure

[5,6], macrophages are also major producers of pro-

inflammatory mediators, and their productive capacity

for these mediators, such as prostaglandin E2, reactive

nitrogen intermediates, interleukin (IL)-6 and tumor

necrosis factor (TNF)-a, is markedly enhanced following

thermal injury [7,8]. There have been several reports

indicating that circulating levels of IL-1b, IL-6 and TNF-a

are increased in patients with burn injury [9].

Leukotrienes, the products generated by the 5-lipox-

ygenase pathway, are particularly important in inflamma-

tion; indeed, leukotrienes increase microvascular

permeability and are potent chemotactic agents [10]. There

are a number of studies demonstrating the role of

leukotrienes as mediators of the gastric damage induced

by ethanol or other noxious substances [11,12]. Cysteinyl

leukotrienes, namely leukotrienes C4, D4 and E4 (LTC4,

LTD4 and LTE4) are secreted mainly by eosinophils, mast

cells, monocytes and macrophages, and they exert a variety

of actions which emphasize their importance as pathogenic

elements in inflammatory states [13,14]. A selective

reversible cys-leukotriene-1 receptor (LTD4 receptor)

antagonist, montelukast (MK-0476), is used in the treatment

of asthma and is reported to reduce eosinophilic inflamma-

tion in the airways [15,16], while leukotriene receptor

antagonists or biosynthesis inhibitors have been reported to

ameliorate ethanol-induced gastric mucosal damage [11,17],

and experimental colitis [18]. Previous studies in burned

patients [19] and experimental studies [20] have shown that

the lipoxygenase metabolite LTB4 and cysteinyl leuko-

trienes are involved in the tissue trauma. However, the

potential effect of montelukast in thermal injury-induced

multiple organ damage was not studied so far.

Based on these findings, in the present study, the putative

protective effect of montelukast against burn-induced skin-

and remote organ-injury was examined using biochemical

and histopathological approaches, while the functional

impairments were monitored by hepatic and renal function

tests.

2. Materials and methods

2.1. Animals

Wistar albino rats of both sexes, weighing 200–250 g,

were obtained from Marmara University School of Medicine

Animal House. The rats were kept at a constant temperature

(22 � 1 8C) with 12-h light:12-h dark cycles, were fed with

standard rat chow and were fasted for 12 h before the

experiments, but were allowed free access to water. All

experimental protocols were approved by the Marmara

University School of Medicine Animal Care and Use

Committee.

2.2. Thermal injury and experimental design

Under brief ether anesthesia, dorsum of the rats was

shaved, exposed to 90 8C water bath for 10 s, which resulted

in partial-thickness second-degree skin burn involving 30%

of the total body surface area. All the animals were then

resuscitated with physiological saline solution (10 ml/kg,

subcutaneously). Montelukast (10 mg/kg) or saline was

administered intraperitoneally immediately after and at 12th

hour of the burn injury. In both saline (burn group) and

montelukast-treated (burn + ML) groups, rats were decapi-

tated at 24 h following burn injury. In order to rule out the

effects of anesthesia, the same protocol was applied in the

control group, except that the dorsums were dipped in a

25 8C water bath for 10 s. Each group consisted of eight rats.

After decapitation, trunk blood was collected, the serum

was separated to measure the aspartate aminotransferase

(AST), alanine aminotransferase (ALT) levels and creati-

nine, blood urea nitrogen (BUN) as indicators of liver and

kidney function, respectively. Lactate dehydrogenase (LDH)

and TNF-a were also assayed in serum samples for the

evaluation of generalized tissue damage.

A dorsal skin area of 3 cm � 3 cm with full-thickness

was carefully excised and weighed (g/cm2). In order to

evaluate the presence of oxidant injury in the skin and distant

organs, tissue samples from the lung, liver, kidney and skin

samples were stored at �80 8C for the determination of

malondialdehyde (MDA) and glutathione levels, myelo-

preoxidase activity and collagen content. For histological

analysis, samples of the tissues were fixed in 10% (v/v)

buffered p-formaldehyde and prepared for routine paraffin

embedding. Tissue sections (6 mm) were stained with

Hematoxylin and Eosin and examined under a light

microscope (Olympus-BH-2). An experienced histologist

who was unaware of the treatment conditions made the

histological assessments.

2.3. Assays

Blood urea nitrogen [21] and serum AST, ALT [22] and

creatinine [23] concentrations and LDH levels [24] were

determined spectrophotometrically using an automated

analyzer. TNF-a was evaluated by a RIA-IRMA (radio-

immunoassay–immunoradiometric assay) method. All sam-

ples were assayed in duplicates using the commercial kit

(Biosource Europe S.A., Nivelles, Belgium). The activity of

radioactive assays was measured by a gamma counter (LKB

WALLAC 1270 RACK Gamma Counter, Canada). TNF-a

in the serum samples was expressed as ng/ml.

Tissue samples were homogenized with ice-cold 150 mM

KCl for the determination of malondialdehyde (MDA) and

G. Sener et al. / Burns 31 (2005) 587–596 589

Fig. 1. (A) Burn-induced increase in the skin wet-weight in saline-treated

burn group compared to control (C), montelukast-treated-control (ML) and

montelukast-treated burn (burn + ML) groups; (B) TNF-a levels in serum

samples of control, montelukast-treated control (ML), saline-treated burn

(burn) and montelukast-treated burn (burn + ML) groups. ***p < 0.001 vs.

control group; ++p < 0.01 and +++p < 0.001 vs. burn group. For each group

n = 8.

glutathione (GSH) levels. The MDA levels were assayed for

products of lipid peroxidation by monitoring thiobarbituric

acid reactive substance formation as described previously

[25]. Lipid peroxidation was expressed in terms of MDA

equivalents using an extinction coefficient of 1.56 � 105 M/

cm and results are expressed as nmol MDA/g tissue. GSH

measurements were performed using a modification of the

Ellman procedure [26]. Briefly, after centrifugation at

3000 rev/min for 10 min, 0.5 ml of supernatant was added to

2 ml of 0.3 mol/l Na2HPO4�2H2O solution. A 0.2 ml

solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium

citrate) was added and the absorbance at 412 nm was

measured immediately after mixing. GSH levels were

calculated using an extinction coefficient of 13600 M/cm.

Results are expressed in mmol GSH/g tissue.

Myeloperoxidase (MPO) is an enzyme that is found

predominantly in the azurophilic granules of polymorpho-

nuclear leukocytes (PMN). Tissue MPO activity is

frequently utilized to estimate tissue PMN accumulation

in inflamed tissues and correlates significantly with the

number of PMN determined histochemically in tissues [27].

MPO activity was measured in tissues in a procedure similar

to that documented by Hillegas et al. [28]. Tissue samples

were homogenized in 50 mM potassium phosphate buffer

(PB, pH 6.0), and centrifuged at 41,400 � g (10 min);

pellets were suspended in 50 mM PB containing 0.5%

hexadecyltrimethylammonium bromide (HETAB). After

three freeze and thaw cycles, with sonication between

cycles, the samples were centrifuged at 41,400 � g for

10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction

mixture containing 50 mM PB, o-dianisidine, and 20 mM

H2O2 solution. One unit of enzyme activity was defined as

the amount of MPO present that caused a change in

absorbance measured at 460 nm for 3 min. MPO activity

was expressed as U/g tissue.

Tissue collagen was measured as a free radical-induced

fibrosis marker. Tissue samples were cut with a razor blade,

immediately fixed in 10% formalin in 0.1 M phosphate buffer

(pH 7.2) in paraffin, and approximately 15 mm thick sections

were obtained. Evaluation of collagen content was based on

the method published by Lopez de Leon and Rojkind [29],

which is based on selective binding of the dyes Sirius Red and

Fast Green FCF to collagen and noncollagenous components,

respectively. Both dyes were eluted readily and simulta-

neously by using 0.1 N NaOH–methanol (1:1, v/v). Finally,

the absorbances at 540 and 605 nm were used to determine the

amount of collagen and protein, respectively.

2.4. Statistics

Statistical analysis was carried out using GraphPad Prism

3.0 (GraphPad Software, San Diego; CA; USA). All data

were expressed as means � S.E.M. Groups of data were

compared with an analysis of variance (ANOVA) followed

by Tukey’s multiple comparison tests. Values of p < 0.05

were regarded as significant.

3. Results

Burn-induced increase in the skin wet-weight, as

compared to control group ( p < 0.001), was abolished in

montelukast-treated group ( p < 0.01)(Fig. 1A). TNF-a

levels were also significantly increased in saline-treated

burn group, while this burn-induced rise in serum TNF-a

level was significantly reversed with montelukast treatment

(Fig. 1B).

BUN and creatinine concentrations were studied to assess

renal function, while serum AST and ALT levels were

determined to evaluate hepatic function. BUN level in the

burn group was found to be significantly higher than that of

the control rats ( p < 0.001), while blood creatinine level,

was not changed after burn (Table 1). When montelukast

was administered following burn injury, elevation in BUN

level was abolished ( p < 0.01). AST and ALT levels were

significantly higher in the burn group when compared with

those of the control group ( p < 0.001). Although mon-

telukast treatment decreased both AST and ALT levels

significantly ( p < 0.001), these parameters were found to be

still significantly higher than the control values. Similarly,

serum LDH activity showed a significant increase in the burn

group, indicating generalized tissue damage, ( p < 0.001),

and this effect was reversed significantly by montelukast

treatment ( p < 0.001; Table 1).

G. Sener et al. / Burns 31 (2005) 587–596590

Table 1

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine and lactate dehydrogenase levels in control,

montelukast-treated control (ML), saline-treated burn (burn) and montelukast-treated burn (burn + ML) groups (n = 8 per group)

Control ML Burn Burn + ML

ALT (mg/dl) 77.3 � 2.2 71.4 � 2.7 168.7 � 8.5*** 106.1 � 6.8*,+++

AST (mg/dl) 181.4 � 6.8 179.7 � 6.6 485.8 � 33.5*** 326.7 � 23.5 ***,+++

BUN (mg/dl) 28.5 � 1.9 28.3 � 1.7 39.7 � 0.9*** 33.0 � 0.6++

Creatinine (mg/dl) 0.45 � 0.1 0.51 � 1.1 0.56 � 0.2 0.46 � 0.1

LDH (U/l) 1013 � 28.9 1062 � 23.8 1668 � 36.7*** 1136 � 39.0***

* p < 0.05.*** p < 0.001; compared with control group.++ p < 0.01.

+++ p < 0.001; compared with saline-treated burn group.

MDA levels determined in the skin, lung, liver and kidney

tissues were found to be significantly higher in the saline-

treated burn group than those in the control group ( p < 0.01

and 0.001), while treatment with montelukast reversed these

elevations back to control levels ( p < 0.01 and 0.001;

Fig. 2). On the other hand, GSH levels in all the studied

tissues were significantly decreased following burn injury

( p < 0.05 and 0.001), while montelukast treatment inhibited

the depletion of GSH stores ( p < 0.05 and 0.001) (Fig. 3). In

the saline-treated burn group, MPO activities in both the skin

and the three distant organs were found to be increased

significantly ( p < 0.01 and 0.001), while treatment with

leukotriene receptor antagonist reversed these elevations

( p < 0.05 and 0.01; Fig. 4). As an indicator of enhanced

Fig. 2. Malondialdehyde (MDA) levels in the lung, liver, kidney and skin samples

and montelukast-treated burn (burn + ML) groups. For each group n = 8. **p < 0.0

tissue fibrotic activity, the collagen contents in skin, lung,

liver and kidney demonstrated significant increases in

saline-treated burn group ( p < 0.05 and 0.01), while

montelukast treatment prevented these alterations (Fig. 5).

Histopathological examination of the skin tissues with

burn wounds confirmed severe loss of the epidermis with

diffuse edema in both the dermis and hypodermis (Fig. 6a),

while the control group demonstrated intact epidermis and

dermis with well-developed keratin layers. In the mon-

telukast-treated burn group, the regular morphology of both

epidermis and dermis layers was apparent and the keratin

layer was maintained as in the control group (Fig. 6b). The

regular structure of the alveoli and interstitium observed in

the control group was replaced with massive alveolar

of control (C), montelukast-treated control (ML), saline-treated burn (burn)

1, ***p < 0.001 vs. control group. ++p < 0.01, +++p < 0.001 vs. burn group.

G. Sener et al. / Burns 31 (2005) 587–596 591

Fig. 3. Glutathione (GSH) levels in the lung, liver, kidney and skin samples of control (C), montelukast-treated control (ML), saline-treated burn (burn) and

montelukast-treated burn (burn + ML) groups. For each group n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. +p < 0.05, ++p < 0.01, +++p < 0.001

vs. burn group.

structural disturbance and infiltration of inflammatory cells

with prominent hemorrhage in the interstitium of the

saline-treated burn group (Fig. 7a). In the montelukast-

treated burn group, interstitial congestion was still present,

Fig. 4. Myeloperoxidase activity (MPO) in the lung, liver, kidney and skin samples

and montelukast-treated burn (burn + ML) groups. For each group n = 8. **p < 0.

burn group.

but the interstitial hemorrhage has disappeared and the

structure of alveoli was organized (Fig. 7b). In the liver

parenchyma of the saline-treated burn group, a severe

hemorrhage, infiltration with inflammatory cells, and a

of control (C), montelukast-treated control (ML), saline-treated burn (burn)

01, ***p < 0.001 vs. control group. +p < 0.05, ++p < 0.01, +++p < 0.001 vs.

G. Sener et al. / Burns 31 (2005) 587–596592

Fig. 5. Collagen content in the lung, liver, kidney and skin samples of control (C), montelukast-treated control (ML), saline-treated burn (burn) and

montelukast-treated burn (burn + ML) groups. For each group n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. +p < 0.05, +++p < 0.001 vs. burn

group.

prominent dilation of the sinusoids with Kupffer cells were

observed (Fig. 8a), while the control group revealed a

regular morphology of liver parenchyma with well-

designated hepatic cells and sinusoids. Although in the

montelukast group, mild hemorrhage in the parenchyma,

aggregations of erythrocytes and increased Kupffer cells

with sinusoidal dilatation were still present in some of the

sinusoids, the general morphology of cells and sinusoids

seemed organized (Fig. 8b). While the control group had a

regular morphology of renal parenchyma with well-

designated glomeruli and tubules, burn-induced renal

Fig. 6. Micrographs of lung tissue. (a) Burn group, massive alveolar structural distu

(arrowheads) in the interstitium; insert: alveoli filled with blood cells. (b) Burn + m

the structure of alveoli was reorganized ( ). HE �200, inset �400.

damage was observed with the presence of severe

hemorrhage in the kidney parenchyma, edema and

hemorrhage in the intertubular interstitium, glomerular

degeneration and cellular debris in the tubuli (Fig. 9a). On

the other hand, in the montelukast-treated group, mild

vasocongestion in the parenchyma and the cellular debris

in the proximal tubules were still persistent, but the severe

hemorrhage was no longer present and the glomeruli

maintained a better morphology (Fig. 9b). Montelukast

treatment in the control group had no impact on the

morphology in any studied tissue (data not shown).

rbance ( ) and infiltration of inflammatory cells with prominent hemorrhage

ontelukast group, mild interstitial congestion was still present (arrowhead),

G. Sener et al. / Burns 31 (2005) 587–596 593

Fig. 7. Micrographs of liver tissue. (a) Burn group, severe hemorrhage and infiltration with inflammatory cells in the liver parenchyme (arrows), central vein ( ),

prominent dilatation of the sinusoids with Kuppfer cells (arrowhead inset), (b) burn + montelukast group, morphology of cells and sinusoids seem reorganised

but sinusoidal dilatation was still present (inset), HE �200, inset �400.

4. Discussion

As evidenced by alterations in malondialdehyde and

glutathione levels, myeloperoxidase activity and collagen

content, the results of the present study demonstrate that the

burn-induced local damage, as well as distant organ injury in

the lung, liver and kidney tissues, is ameliorated by

montelukast treatment. Moreover, morphological changes

in the injured tissues and impairments in hepatic and renal

functions due to burn trauma were also improved by

montelukast treatment, which also reduced the serum levels

of the proinflammatory cytokine TNF-a. These findings

suggest that montelukast, by inhibiting neutrophil infiltra-

tion and subsequent activation of inflammatory mediators

that induce lipid peroxidation, appears to have a protective

role in the burn-induced oxidative injury of the skin and the

involved organs distant to the original wound.

Fig. 8. Micrographs of kidney tissue. (a) Burn group, severe hemorragia in the ki

structure of the glomeruli showed degeneration (arrows), there was cellular debris i

morphology ( ) and severe hemorrhage was no longer present. HE �200.

The local and systemic inflammatory responses to severe

burn are extremely complex, resulting in both local damage

and marked systemic effects. Much of the local and certainly

the majority of the distant changes are caused by

inflammatory mediators [30]. Generalized tissue inflamma-

tion is present in injured organs within hours of injury, even

in the absence of shock. On the other hand, circulating

endotoxins, which become evident probably as a result of

burn wound colonization and an early gut leak, lead to the

activation of the macrophages and neutrophils [31]. In

addition to xanthine oxidase-related free radical generation

in burn trauma, adhesion of leukocytes to endothelial cells,

which consequently are the targets for leukocyte products,

activates the neutrophils to produce additional free radicals.

It has been shown that post-burn intravascular haemolysis

and lung injury are prevented by neutrophil depletion of

experimental animals, verifying the role of neutrophils in the

dney parenchyme, edema and hemorrhage the intertubular interstitium, the

n the tubuli. (b) Burn + montelukast group, the glomeruli maintained a better

G. Sener et al. / Burns 31 (2005) 587–596594

Fig. 9. Micrographs of skin tissue. (a) Burn group, severe loss of the epidermis ( ) with diffuse edema and congestion (insert, arrowhead) in the dermis. (b)

Burn + montelukast group, regular morphology of both epidermis ( ) and dermis layers was apparent and the keratine layer was maintained as the control group,

note the regenerated hair follicle structure (arrowhead). HE �200, inset �400.

development of remote organ injury [32–34]. The over-

production of highly reactive metabolites, such as oxygen

free radicals, initiates a cascade of inflammatory processes

in thermally damaged skin, leading to enhanced tissue loss

and delayed wound healing [35]. In the presence of

neutrophil-derived MPO, reactive oxygen products can

generate hypocholorus acid (HOCl) and initiate the

deactivation of anti-proteases and activation of latent

proteases, leading to tissue damage [36]. MPO activity,

which is used as an indirect evidence of tissue neutrophil

infiltration, was increased in the lung, hepatic and renal

tissues, while tissue MPO levels were significantly

decreased with montelukast treatment, suggesting that the

protective effect of montelukast in burn-induced oxidative

injury involves, in part, the inhibition of neutrophil

infiltration to the skin, as well as to the tissues distant to

original burn trauma.

Macrophages are also major producers of pro-inflam-

matory mediators and the productive capacity of macro-

phages for inflammatory mediators (i.e., nitric oxide,

prostaglandins, TNF-a, IL-6, etc.) is profoundly increased

post-burn [2,7,37]. TNF-a and TNF receptor levels were

shown to reflect the burn severity and outcome of the burns

[38]. In accordance with these findings, the present results

demonstrate that burn trauma increases serum TNF-a level,

indicating the role of this cytokine in burn-induced local and

systemic inflammation. On the other hand, blockade of

leuktriene receptors with montelukast depressed the TNF-a

response. Since the inhibition of 5-lipoxygenase was shown

to reduce the expression of TNF-a [39], it seems likely that

the anti-inflammatory effect of montelukast in burn injury

involves the suppression of a variety of pro-inflammatory

mediators produced by the leukocytes and macrophages.

It has been suggested that increased synthesis of

peptidoleukotrienes may occur in various inflammatory

diseases, including burns. Struck et al. treated chemically

burned eyes of the rabbit with S872419, a specific receptor

antagonist of peptide leukotrienes, and suggested that the

inhibition of lipoxygenase-mediated reactions would

improve the anti-inflammatory drug therapy after burn

[40]. It was also reported that patients with severe injuries

excrete higher levels of urinary LTE4 than healthy

volunteers [41]. Moreover, patients with lung injury have

elevated airway leukotriene levels, reflecting airway

epithelial damage and sulfidopeptide leukotrienes (LTC4/

D4/E4) are suspected to be important lipid mediators in

inflammatory responses in the lung [42].

It is clear that after burn trauma, tissue adenosine

triphosphate (ATP) levels gradually fall, and increased

adenosine monophosphate (AMP) is converted to hypox-

anthine, providing substrate for xanthine oxidase [2]. These

complicated reactions produce hydrogen peroxide and

superoxide, clearly recognized deleterious free radicals.

Free radical-mediated cell injury has been supported by

post-burn increases in systemic and tissue levels of lipid

peroxidation products such as conjugated dienes, thiobarbi-

turic acid reaction products, or malondialdehyde levels

[2,43]. Damage of membranes by lipid peroxidation and by

exposure to mediators such as platelet activating factor,

leukotrienes and proteases, leads to increased permeability,

tissue oedema and organ dysfunction. In the present study,

injury caused significant elevations in MDA levels both

locally and in the distant tissues of burned animals, while

glutathione levels were depressed concomitantly. The

relationship between the amount of products of oxidative

metabolism and natural scavengers of free radicals

determines the outcome of local and distant tissue damage

and further organ failure in burn injuries [43]. Since

antioxidants and other agents that control phagocyte

function are likely to contribute to the protection of the

tissues against burn, it is conceivable that antioxidant

therapy (glutathione, N-acetyl-L-cysteine, or Vitamins A, E

and C) reduces tissue lipid peroxidation, burn and burn/

sepsis-mediated mortality, attenuates changes in cellular

G. Sener et al. / Burns 31 (2005) 587–596 595

energetics and maintains microvascular circulation

[33,44–48]. Reduced thiol agents, such as GSH, which

are capable of interacting with free radicals to yield more

stable elements, are known for their ability to repair

membrane lipid peroxides [49]. As reported by Ross, cell

injury and enhanced cell susceptibility to toxic chemicals

are related to the efflux of GSH precurcors and hence to

diminished GSH biosynthesis [50]. In this sense, GSH and

other antioxidants play a critical role in limiting the

propagation of free-radical reactions, which would

otherwise result in extensive lipid peroxidation. In the

present study, thermal trauma significantly depleted tissue

GSH stores, indicating that GSH was used as an

antioxidant for the detoxification of toxic oxygen

metabolites, while the susceptibility of the involved

tissues to oxidative injury was enhanced. Due to its

antioxidant activity, montelukast treatment reduced the

burn-induced oxidative injury and restored the GSH levels

significantly. These data collectively support the hypoth-

esis that cellular oxidative stress is a critical step in burn-

mediated injury, and suggest that antioxidant strategies

designed either to scavenge free radicals, or to inhibit free

radical formation and leukotriene production may provide

organ protection in patients with burn injury.

Despite recent advances, multiple organ failure (such as

cardiac instability, respiratory, hepatic or renal failure) and

compromised immune function, which results in increased

susceptibility to subsequent sepsis, remain major causes of

burn morbidity and mortality [7]. The findings of the current

study illustrate that leukotriene receptor antagonist reverses

systemic inflammatory reaction to thermal trauma, and

thereby reduces multiple organ failure, implicating the role

of leukotrienes in the pathogenesis of burn-induced

inflammation and multiple organ failure. These data suggest

that montelukast may be of therapeutic use in preventing

burn-induced local inflammation and multiple organ

dysfunction, by depressing neutrophil infiltration and the

release of proinflammatory cytokines. Thus, the antioxidant

and anti-inflammatory effects of montelukast might be

applicable to clinical situations to ameliorate burn-induced

multiple organ failure.

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