2-Mercaptoethane sulfonate (MESNA) protects against burn-induced renal injury in rats

Burns 30 (2004) 557–564

2-Mercaptoethane sulfonate (MESNA) protects against burn-induced renal injury in rats a,∗ , Özer Sehirli a , Gözde Erkanlı b , Sule Göksel Sener ¸ ¸ Cetinel b , Nursal Gedik c , Berrak Ye˘gen d b

a Marmara University, School of Pharmacy, Department of Pharmacology, Tıbbiye Cad. 34668 Istanbul, ˙ Turkey ˙ Marmara University, School of Medicine, Departments of Histology–Embryology, Tıbbiye Cad. 34668 Istanbul, Turkey c Kasımpasa Military Hospital, Division of Biochemistry; Istanbul, ˙ Turkey d Marmara University, Department of Physiology, Istanbul, ˙ Turkey

Accepted 16 February 2004

Abstract Animal models of thermal injury implicate oxygen radicals as causative agents in local wound response and distant organ injury following burn. In this study we investigated the putative protective effects of 2-mercaptoethane sulfonate (MESNA) against oxidative kidney damage in rats with thermal injury. Under ether anaesthesia, shaved dorsum of the rats was exposed to 90 ◦ C bath for 10 s to induce burn injury. Rats were decapitated either 6 or 24 h after burn injury. MESNA was administered i.p. immediately after burn injury. MESNA injections were repeated once more 12 h after the first injection in the 24 h burn group. In the control group the same protocol was applied except that the dorsum was dipped in a 25 ◦ C water bath for 10 s. Kidney tissues were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, protein oxidation (PO), myeloperoxidase (MPO) activity and collagen contents. Creatinine, urea concentrations (BUN) and lactate dehydrogenase (LDH) in blood were measured for the evaluation of renal functions and tissue damage, respectively. Tissues were also examined microscopically. Severe skin scald injury (30% of total body surface area) caused significant decrease in GSH level, significant increase in MDA level, protein oxidation (PO), MPO activity and collagen content of renal tissue. Serum creatinine was slightly increased at the early phase of thermal trauma but not changed in 24 h groups. On the other hand BUN and LDH were significantly elevated by thermal trauma in both 6 and 24 h of burn groups. Treatment of rats with MESNA significantly increased the GSH level and decreased the MDA level, PO, MPO activity, collagen contents, BUN and LDH. Since MESNA reversed the oxidant responses seen in burn injury, it seems likely that MESNA could protect against thermal trauma-induced renal damage. © 2004 Elsevier Ltd and ISBI. All rights reserved. Keywords: MESNA; Burn; Lipid peroxidation; Glutathione

1. Introduction The inflammatory response to thermal injury is extremely complex, resulting in local tissue damage and deleterious systemic effects in all the organ systems distant from the original wound. Major thermal injury induces the activation of an inflammatory cascade that contributes to the development of subsequent immunosuppression, increased susceptibility to sepsis and multiple organ failure [1]. The inflammatory response syndrome in shock-like states might frequently be accompanied by oxidative cell/tissue damage in one or more organ systems in the body [2,3]. Several studies have demonstrated that burn injury is associated with oxygen radical-induced lipid peroxidation, which ∗ Corresponding author. Tel.: +90-216-414-29-62; fax: +90-216-345-29-52. E-mail address: [email protected] (G. Sener). ¸

0305-4179/$30.00 © 2004 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2004.02.008

is an autocatalytic mechanism leading to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic, reactive metabolites and cell death [4,5]. The role of free oxygen radicals in ischemic insult or organ failure is well documented [6,7]. The inflammatory response, which leads to hyperactivation of tissue neutrophils could contribute to oxidative cell/tissue damage [2]. Macrophages are also major producers of pro-inflammatory mediators and their productive capacity for these mediators is markedly enhanced following thermal injury [8]. Thus, it appears that tissue injury after thermal trauma is mediated by both reactive oxygen metabolites (ROM) and activated neutrophils and macrophages [9–11]. Treatment approaches to major burn injury include administration of crystalloid solutions to correct hypovolemia and to restore peripheral perfusion; however, this aggressive post-burn volume replacement increases oxygen delivery to previously ischemic tissue. Thus, the restoration of

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oxygen delivery is thought to initiate a series of deleterious events that exacerbate ischemia-related tissue injury [12]. It has been shown that antioxidants or free radical scavengers when given post-burn, exert protective effects against thermal trauma-induced oxidative tissue damage and multiple organ failure [13–15]. 2-Mercaptoethane sulfonate (MESNA) is a synthetic small molecule that has the potential to scavenge ROM by virtue of its sulfhydryl group [16]. MESNA was proven to be effective as an antioxidant drug in various in vivo and in vitro models [17]. It is widely used as a systemic protective agent against the toxicity of chemotherapy and is primarily used to reduce hemorrhagic cystitis induced by cyclophosphamide [18]. In addition, MESNA was shown to inhibit the development of bladder tumor in rats [19], and by increasing the kidney levels of free thiol levels it was shown to prevent renal oxidative damage in rats treated with ferric nitrilotriacetate [20]. Owing to its direct suppressive effect on the production of hydrogen peroxide, thiol-containing MESNA may be considered as an antioxidant drug to limit the toxic effects of free radicals produced by all kinds of oxidative injuries. In the present study, the putative protective effect of MESNA against thermal trauma-induced oxidative kidney damage was examined using biochemical approaches; such as measurement of various biochemical parameters like renal malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content as well as renal function tests, and histopathological analysis of renal injury. 2. Materials and methods 2.1. Animals Wistar albino rats of both sexes, weighing 200–250 g, were fasted for 12 h, but were allowed free access to water before experiments. Rats were kept in a room at a constant temperature 22 ± 2 ◦ C with 12 h light and 12 h dark cycles, in individual wire-bottomed cages and fed standard rat chow. All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee. 2.2. Thermal injury Under brief ether anesthesia, dorsum of the rats was shaved, exposed to 90 ◦ C 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, s.c.). Rats were decapitated at 6 or 24 h after burn injury in both vehicle (burn 2 h + saline and burn 24 h + saline) and MESNA-treated (burn 2 h + MESNA and burn 24 h + MESNA) groups. MESNA

(Eczacibasi-BAXTER; 150 mg/kg) or saline was administered intraperitoneally immediately after burn injury and the injections were repeated at the 12th hour in the 24 h burn groups. Each group consisted of eight rats. 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 ◦ C water bath for 10 s. 2.3. Biochemical analysis After decapitation, trunk blood was collected, the serum was separated to measure the creatinine [21], blood urea nitrogen (BUN) [22] and lactate dehydrogenase (LDH) [23] concentrations for the evaluation of renal functions and tissue damage, respectively. Tissue samples from the kidneys were obtained for biochemical and histological analysis. Malondialdehyde levels, an end product of lipid peroxidation; glutathione, a key antioxidant; and protein carbonyl concentration, a specific marker of oxidative damage of proteins were measured in these samples. Tissue-associated myeloperoxidase activity, as an indirect evidence of neutrophil infiltration, and collagen content as a free radical-induced fibrosis marker were also measured in all tissue samples. 2.4. Malondialdehyde and glutathione assays Tissue samples were homogenized with ice-cold 150 mM KCl for the determination of malondialdehyde and glutathione levels. The MDA levels were assayed for products of lipid peroxidation [24]. Results were expressed as nmol MDA/g tissue. Glutathione was determined by the spectrophotometric method, which is based on the use of Ellman’s reagent [25]. Results were expressed ␮mol GSH/g tissue. 2.5. Protein oxidation The protein content of tissue samples was determined by the Lowry assay [26]. Oxidized protein was quantified using the interaction between dinitrophenylhydrazine (DNP) and the carbonyls to yield a chromophore that absorbs strongly at 360 nm [27]. All samples were diluted to 2–4 mg/ml of protein with wash buffer and treated with 1% streptomycin. The carbonyl content was calculated assuming a molar extinction coefficient of 22,000. 2.6. Myeloperoxidase activity Myeleperoxidase activity was measured in tissues in a procedure similar to that documented by Hillegass 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 cy-

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cles, 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 H2 O2 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.

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2.9. 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.

2.7. Tissue collagen measurement Tissue samples were cut with a razor blade, immediately fixed in 10% formaldehyde and then they were embedded in 0.1 M paraffin to obtain approximately 15 ␮m thick sections. The method of collagen measurement is based on the selective binding of Sirius red and Fast Green FCF to collagen and non-collagenous components, respectively, when the sections are stained with both dyes dissolved in aqueous saturated picric acid [29]. Both dyes were eluted readily and simultaneously with NaOH-methanol and the absorbances obtained at 540 and 605 nm were used to determine the amount of the collagen and protein. 2.8. Histological analysis Following the decapitation of rats, small pieces of kidney samples were placed in 10% (v/v) formaldehyde solution and were processed routinely by embedding in paraffin. Tissue sections (6 ␮m) 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.

3. Results 3.1. Renal function tests BUN levels in the both 6 and 24 h burn groups were found to be significantly higher than those in the control rats (P < 0.001; Table 1). When MESNA was administered following burn injury, these levels were significantly reduced (P < 0.001, P < 0.05). Blood creatinine levels, which were elevated only in the 6 h burn group (P < 0.05), were also reduced (P < 0.05) by MESNA treatment. LDH was found significantly higher in both 6 and 24 h burn groups, while MESNA treatment reduced these elevations significantly (P < 0.05–0.001). 3.2. Glutathione (GSH) levels When compared to control group, the levels of renal GSH were decreased at the 6th and 24th hours following burn injury (P < 0.001). MESNA abolished GSH depletion and the measured GSH levels were found to be at the control levels (Fig. 1).

Table 1 Blood urea nitrogen (BUN), serum creatinine and LDH levels in saline- or MESNA-treated animals with burn injury. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001: compared with control group, + P < 0.05, ++ P < 0.01, +++ P < 0.001: MESNA treated groups compared with respective saline treated groups Control BUN (mg/dl) Creatinine (mg/dl) LDH (U/l)

27.3 ± 2.3 0.40 ± 0.02 1618 ± 92

Burn 6 h + saline 3.6∗∗∗

52.0 ± 0.51 ± 0.03∗ 2987 ± 283

Burn 6 h + MESNA 1.5+++

30.8 ± 0.41 ± 0.02+ 1706 ± 137

Burn 24 h + saline 3.1∗∗∗

47.7 ± 0.42 ± 0.02 3513 ± 280

Fig. 1. Glutathione (GSH) levels in the kidney of rats in MESNA or saline-treated groups at 6 and 24 h following burn injury. with control group. +++ P < 0.001: MESNA-treated groups compared with respective saline-treated groups.

Burn 24 h + MESNA 34.3 ± 2.1+ 0.35 ± 0.02 2057 ± 215

∗∗∗ P

< 0.001: compared

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3.3. Malondialdehyde (MDA) levels The kidney MDA levels were found to be significantly higher in both 6 and 24 h burn groups than those in the control group (P < 0.001). Treatment with MESNA significantly reduced the elevations in MDA and reversed back to the control levels (P < 0.001; Fig. 2a). 3.4. Protein oxidation levels In both 6 and 24 h post-burn injury groups, there were significant (P < 0.01–0.001) increases in protein oxidation levels when compared with the control group. MESNA treatment significantly abolished the elevations in protein oxidation at both time points after burn injury (P < 0.01–0.05; Fig. 2b). 3.5. Myeloperoxidase (MPO) activity In both 6 and 24 h burn groups, MPO activity was found to be increased significantly (P < 0.01–0.001), when compared to control group, while MESNA treatment reversed these elevations (P < 0.05–0.01; Fig. 3).

3.6. Renal collagen content and histopathological analysis of tissue injury In the renal tissues, burn-induced significant increases in the collagen content (P < 0.001) were prevented by MESNA treatment (Fig. 4). Light microscopic evaluation of control group had a regular morphology of renal parenchyme (Fig. 5a) with well designated glomeruli and tubules. In the 6 h burn group, there was severe hemorrhage in the kidney parenchyme dominantly in the glomeruli, intertubular interstitium showed edema and there was cellular debris in the proximal tubuli (Fig. 5b). In the MESNA-treated 6 h burn group, mild vasocongestion in the parenchyme persisted but severe hemorrhage was no longer present. The cellular debris in the proximal tubules was still present and the glomeruli maintained a better morphology (Fig. 5c) when compared with burn group. In the 24 h burn group, besides severe congestion of parenchyme, degeneration of both tubules and glomeruli, interstitial edema were observed (Fig. 5d). In most areas the glomeruli had lost their structural morphology and hemorrhage was present. In the MESNA-treated 24 h burn group; congestion and hemorrhage of the parenchyme were no longer observed. The kid-

Fig. 2. (a) Malondialdehyde (MDA), (b) Protein oxidation levels in the kidney of rats in MESNA or saline-treated groups at 6 and 24 h following burn injury. ∗∗ P < 0.01,∗∗∗ P < 0.001: compared with control group. + P < 0.05, ++ P < 0.01, +++ P < 0.001: MESNA-treated groups compared with respective saline-treated groups.

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Fig. 3. Myeloperoxidase (MPO) activities of the kidney in MESNA or saline-treated groups at 6 and 24 h following burn injury. ∗∗ P < 0.01,∗∗∗ P < 0.001: compared with control group. + P < 0.05, ++ P < 0.01, MESNA-treated groups compared with respective saline-treated groups.

Fig. 4. Collagen content in the kidney of rats in MESNA or saline-treated groups at 6 and 24 h following burn injury. ∗ P < 0.05,∗∗∗ P < 0.001: compared with control group. + P < 0.05, +++ P < 0.001: MESNA-treated groups compared with respective saline-treated groups.

ney morphology maintained its integrity whereas there was still minimal persisting cellular debris in the tubuli (Fig. 5e).

4. Discussion The results of the present study demonstrate that burn-induced renal injury, as evidenced by the changes in glutathione and malondialdehyde levels, protein oxidation and myeloperoxidase activity, is ameliorated by MESNA treatment. These findings suggest that MESNA has a protective role in the burn-induced oxidative injury of the kidneys, which may be attributed to its antioxidant effects. Burn shock is a complex process of circulatory and microcirculatory dysfunction, not easily or fully repaired solely by fluid resuscitation. Hypovolemic shock and tissue trauma result in the formation and release of local and systemic mediators, which produce an increase in vascular permeability or an increase in microvascular hydrostatic pressure [30]. It is clear that after burn trauma, tissue adenosine triphosphate (ATP) levels gradually fall, and increased adenosine monophosphate (AMP) is converted to hypoxanthine, providing substrate for xanthine oxidase. These com-

plicated reactions produce hydrogen peroxide and superoxide, clearly recognized deleterious free radicals. In addition to xanthine oxidase-related free radical generation in burn trauma, adherent-activated neutrophils also produce additional free radicals. This enhanced free radical production is paralleled by impaired antioxidant mechanisms, as indicated by burn-related decreases in superoxide dismutase, catalase, glutathione, alpha tocopherol, and ascorbic acid levels [12]. 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, thiobarbituric acid reaction products, or malondialdehyde levels [12]. Antioxidant therapy in burn therapy (ascorbic acid, glutathione, N-acetyl-l-cysteine, or Vitamins A, E, and C alone or in combination) has been shown to reduce burn and burn/sepsis-mediated mortality, attenuate changes in cellular energetics, protect microvascular circulation, reduce tissue lipid peroxidation, improve cardiac output, and to reduce the volume of fluid required in resuscitation [31–36]. In the present study, burn injury caused significant elevations in MDA levels at both 6 and 24 h of burn injury, while glutathione levels were depressed concomitantly in both periods of burn. Reduced thiol agents, such as GSH, capable of

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Fig. 5. (a) Control group, regular structure of glomeruli (arrowheads) and tubuli (arrows), (b) Saline-treated 6 h burn group, severe hemorrhage in the intertitium (arrow) and degenerated glomeruli (arrowheads), (c) MESNA-treated 6 h burn group, decreased interstitial edema and maintained morphology of glomerulus can be observed (arrow), regenerated tubuli with cellular debris, (d) Saline-treated 24 h burn group, interstitial hemorrhage (arrows) and degenerated glomeruli (arrowheads) note that urinary space of glomerulus is lost because of the congestion, (e) MESNA-treated 24 h burn group, regenerated tubuli and glomerulus with urinary space (arrowhead) can be observed, interstitial edema is no longer present, however there is mild congestion still present (arrows).

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interacting with free radicals to yield more stable elements, require close attention for their ability to repair membrane lipid peroxides. In the present study, thermal trauma significantly depleted renal GSH stores, indicating that GSH is 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, MESNA treatment reduced the burn-induced oxidative injury and restored the GSH levels significantly. 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 [37]. 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. On the other hand, cellular proteins are also believed to be a target of oxidative injury. That is, accumulation of oxidized proteins can impair cell function and eventually lead to cell damage. In our study, protein oxidation, which is an early sign of cellular injury, was also reduced by MESNA treatment. These data collectively support the hypothesis that cellular oxidative stress is a critical step in burn-mediated injury, and suggest that antioxidant strategies designed to either inhibit free radical formation or to scavenge free radicals may provide organ protection in patients with burn injury. It has been shown that MESNA, which is rapidly oxidized to the MESNA disulfide form (DIMESNA) in the plasma, can be reduced back to its active thiol form MESNA by the cytosolic enzymes in the renal tubular epithelia. When MESNA is administered at high doses, auto-oxidation process will be saturated and a higher plasma concentration of free MESNA will be presented to the renal tubules. Following its absorption by the tubular cells, this substance will supply free thiol groups, which will continue the detoxification of toxic oxygen metabolites [38]. Thus, the protective effect of MESNA on the kidneys, as observed in the present study, may be attributed to its thiol-supplying action. In an in vitro study, it was shown that hyperthermia-induced cytotoxicity in Chinese hamster ovary cells was reduced and intracellular glutathione levels were increased by MESNA pretreatment [39]. MESNA is primarily used as a chemoprotector agent to reduce hemorrhagic cystitis induced by oxazaphosphorine (e.g. cyclophosphamide and ifosfamide) [40]. Methotrexate-induced cytotoxicity was also limited with MESNA by inhibiting excessive hydrogen peroxide production during chemotherapy [16]. It has been shown that MESNA protects against ferric nitriloacetate [20] or ischemia/reperfusion induced oxidative renal damage [41,42]. Taken together with aforementioned studies, the results of the present study demonstrate that MESNA, by its antioxidant and free radical scavenging ability, ameliorates burn-induced renal dysfunction and associated pathological changes. The local and systemic inflammatory response to thermal injury is extremely complex, resulting in both local damage and subsequent edema, as well as marked systemic effects

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involving organs distant from the burn area itself. Generalized tissue inflammation is present in injured organs within hours of injury, even in the absence of shock. The productive capacity of macrophages for inflammatory mediators (i.e., nitric oxide, prostaglandins, TNF-alpha, IL-6, etc.) is profoundly increased post-burn, implicating that macrophages are involved in the development of the post-burn immunosuppression [12,30]. In addition, tissue injury after thermal trauma is also mediated by activated neutrophils. It has also been shown that post-burn intravascular haemolysis, as well as the onset of lung injury after thermal trauma of the skin, were largely prevented by neutrophil depletion of experimental animals, suggesting that neutrophils play an important role in the development of remote organ injury [15,34,43]. Activated neutrophils, lead to the formation of toxic oxygen products, which further cause tissue damage. Reactive oxygen products can generate hypocholorus acid (HOCl) in the presence of neutrophil-derived myeloperoxidase and initiate the deactivation of antiproteases and activation of latent proteases, leading to tissue damage [44]. MPO activity is used as an indirect evidence of neutrophil infiltration in oxidative tissue injury. Our observation, showing increased MPO levels in the renal tissues, indicates that neutrophil accumulation in the kidneys contributes to distant organ injury. Since renal MPO levels were significantly decreased after MESNA treatment, the results also suggest that the protective effect of MESNA in burn-induced renal injury involves, in part, the inhibition of neutrophil infiltration to the injured tissue distant to original burn trauma. Despite recent advances, multiple organ failure remains a major cause of burn morbidity and mortality. The findings of the current study illustrate that MESNA, as an antioxidant and thiol-containing drug, protects renal tissue against burn-induced oxidative injury by a neutrophil-dependent mechanism. These findings raise the possibility that MESNA can be regarded as a potential agent in limiting burn-induced renal complications. References [1] Schwacha MG, Chaudry IH. The cellular basis of post-burn immunosuppression: macrophages and mediators. Int J Mol Med 2002;10:239–43. [2] Sayeed MM. Neutrophil signaling alteration: an adverse inflammatory response after burn shock. Medicina (B Aires) 1998;58:386–92. [3] Carsin H, Bargues L, Stephanazzi J, Paris A, Aubert P, Le Bever H. Inflammatory reaction and infection in severe burns. Pathol Biol (Paris) 2002;50(2):93–101. [4] Demling RH, LaLonde C. Systemic lipid peroxidation and inflammation induced by thermal injury persists into the post resuscitation period. J Trauma 1990;30:69–74. [5] Eschwege P, Paradis V, Conti M, Holstege A, Richet F, Deteve J, et al. In situ detection of lipid peroxidation by-products as markers of renal ischemia injuries in rat kidneys. J Urol 1999;162:553–7. [6] Ward PA, Till GO. Pathophysiologic events related to thermal injury of skin. J Trauma 1990;30(12 Suppl):S75–9. [7] Youn Y-K, LaLonde C, Demling R. The role of mediators in the response to thermal injury. World J Surg 1992;16:30–6.

564

G. S¸ ener et al. / Burns 30 (2004) 557–564

[8] Schwacha MG. Macrophages and post-burn immune dysfunction. Burns 2003;29:1–14. [9] Mileski W, Borgstrom D, Lightfoot E, Rothlein R, Faanes R, Lipsky P, et al. Inhibition of leukocytes-endothelial adherences following thermal injury. J Surg Res 1992;52:334–9. [10] Hatheril JR, Till GO, Bruner LH, Ward PA. Thermal injury, intravascular hemolysis, and toxic oxygen products. J Clin Invest 1986;78:629–36. [11] Hansbrough JF, Wikström T, Braide M, Tenenhaus M, Rennekampff OH, Kiessig V, et al. Neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury. J Surg Res 1996;61:17– 22. [12] Horton JW. Free radicals and lipid peroxidation mediated injury in burn trauma: the role of antioxidant therapy. Toxicology 2003;189:75–88. [13] Sener ¸ GA, Sehirli ¸ AO, Satıro˘ ¸ glu H, Keyer-Uysal M, Ye˘gen CB. Melatonin improves oxidative organ damage in a rat model of thermal injury. Burns 2002;28:419–25. [14] Çetinkale O, Senel O, Bulan R. The effect of antioxidant therapy on cell-mediated immunity following burn injury in an animal model. Burns 1999;25:113–8. [15] Horton JW, White DJ, Maass DL, Hybki DP, Haudek S, Giroir B. Antioxidant vitamin therapy alters burn trauma-mediated cardiac NF-kappaB activation and cardiomyocyte cytokine secretion. J Trauma 2001;50:397–408. [16] Gressier B, Lebegue S, Brunet C, Luyckx M, Dine T, Cazin M, et al. Pro-oxidant properties of methotrexate: evaluation and prevention by anti-oxidant drug. Pharmazie 1994;49:679–81. [17] Gressier B, Lebegue N, Brunet C, Luyckx M, Dine T, Cazin M, et al. Scavenging of reactive oxygen species by letosteine, a molecule with two blocked-SH groups. Comparison with free –SH drugs. Pharm World Sci 1995;17:76–80. [18] Berrigan MJ, Marinello AJ, Pavelic Z, Williams CJ, Struck RF, Gurtoo HL. Protective role of thiols in cyclophosphamide-induced urotoxicity and depression of hepatic drug metabolism. Cancer Res 1982;42:3688–95. [19] Nishikawa A, Morse MA, Chung F-L. Inhibitory effects of 2-mercaptoethane sulfonate and 6-phenylhexyl isothiocyanate on urinary bladder tumorigenesis in rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. Cancer Lett 2003;193:11–6. [20] Umemura T, Hasegawa R, Sai-Kato K, Nishikawa A, Furukawa F, Toyokuni S, et al. Prevention by 2-mercaptoethane sulfonate and N-acetylcysteine of renal oxidative damage in rats treated with ferric nitrilotriacetate. Jpn J Cancer Res 1996;87:882–6. [21] Slot C. Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest 1965;17:381–7. [22] Talke H, Schubert GE. Enzymatic urea determination in the blood and serum in the Warburg optical test. Klin Wochenschr 1965;43:174–5. [23] Martinek RG. A rapid ultraviolent spectrophotomeetric lactic dehydrogenase assay. Clin Chem Acta 1972;40:91–9. [24] Beuge JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302–11. [25] Beutler E. Glutathione in red blood cell metabolism. A Manuel of Biochemical Methods. Grune & Stratton: New York; 1975. p. 112–4.

[26] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [27] Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz A, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464–78. [28] Hillegass LM, Griswold DE, Brickson B, Albrightson-Winslow C. Assessment of myeloperoxidase activity in whole rat kidney. J Pharmacol Methods 1990;24:285–95. [29] Lopez De Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed parraffin-embedded sections. J Histochem Cytochem 1985;33:737–43. [30] Warden GD, Heimbach DM. Burns. In: Principles of Surgery. Schwartz SI, editor, vol. 1. McGraw-Hill Companies Inc.; 1999. p. 223–95. [31] Choi M, Ehrlich HP. U75412E, a lazaroid, prevents progressive burn ischemia in a rat burn model. Am J Pathol 1993;142:519–28. [32] Thomson PD, Till GO, Smith Jr DJ. Modulation of IgM antibody formation by lipid peroxidation products from burn plasma. Arch Surg 1991;126:973–6. [33] Tanaka H, Lund T, Wiig H, Reed RK, Yukioka T, Matsuda H, et al. High dose vitamin C counteracts the negative interstitial fluid hydrostatic pressure and early edema generation in thermally injured rats. Burns 1999;25:569–74. [34] LaLonde C, Nayak U, Hennigan J, Demling RH. Excessive liver oxidant stress causes mortality in response to burn injury combined with endotoxin and is prevented with antioxidants. J Burn Care Rehabil 1997;18:187–9. [35] Konukoglu D, Cetinkale O, Bulan R. Effects of N-acetylcysteine on lung glutathione levels in rats after burn injury. Burns 1997;23:541–4. [36] Kanda Y, Yamamoto N, Yoshino Y. Utilization of vitamin A in rats with inflammation. Biochim Biophys Acta 1990;1034:337–41. [37] Ross D. Glutathione, free radicals and chemotherapeutic agents. Pharm Ther 1988;37:231–49. [38] Skinner R, Sharkey IM, Pearson ADJ, Craft AW. Ifosfamide, mesna, and nephrotoxicity in children. J Clin Oncol 1993;11:173–90. [39] Lord-Fontaine S, Averill DA. Enhancement of cytotoxicity of hydrogen peroxide by hyperthermia in chinese hamster ovary cells: role of antioxidant defenses. Arch Biochem Biophys 1999;363:283– 95. [40] Souid A-K, Fahey RC, Aktas MK, Sayın OA, Karjoo S, Newton GL, et al. Blood thiols following amifostine and mesna infusions, a pediatric oncology group study. Drug Metab Dispos 2001;29:1460–6. [41] Cargnoni A, Comini L, Boraso A, De Giuli F, Scotti C, Ferrari R. The effects of l-arginine mono(2-mercaptoethanesulfonate) on the ischemic and reperfused heart. Cardioscience 1992;3:179–87. [42] Mashiach E, Sela S, Weinstein T, Cohen HI, Shasha SM, Kristal B. Mesna: a novel renoprotective antioxidant in ischaemic acute renal failure. Nephrol Dial Transplant 2001;16:542–51. [43] Khodr B, Khalil Z. Modulation of inflammation by reactive oxygen species: implacations for aging and tissue repair. Free Radic Biol Med 2001;30:1–8. [44] Kettle AJ, Winterbourn CC. Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Rep 1997;3:3–15.