www.elsevier.com/locate/jpedsurg

Journal of Pediatric Surgery (2011) 46, 1746–1752

Mercaptoethylguanidine attenuates caustic esophagealinjury in rats: a role for scavenging of peroxynitriteAhmet Guven a,⁎, Bulent Uysal b, Bahadir Caliskan a, Emin Oztas c,Haluk Ozturk a, Ahmet Korkmaz b

aDepartment of Pediatric Surgery, Gulhane Military Medical Academy, 06170 Etlik, Ankara, TurkeybDepartment of Physiology, Gulhane Military Medical Academy, 06170 Etlik, Ankara, TurkeycDepartment of Histology and Embrology, Gulhane Military Medical Academy, 06170 Etlik, Ankara, Turkey

Received 22 November 2010; revised 11 February 2011; accepted 13 February 2011

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Key words:Caustic esophageal burn;Mercaptoethylguanidine;Antioxidant enzymes;Scavenging of peroxinitrate

AbstractIntroduction: After ingestion of caustic material, tissue damage is caused by reactive oxygen speciesand reactive nitrogen species such as peroxynitrite. Mercaptoethylguanidine (MEG) is a well-knownscavenger of peroxynitrite. This study was designed to determine whether MEG has a beneficial effecton caustic esophageal injury.Materials and Methods: Forty-five rats were allocated into 3 groups: sham-operated, untreated, andtreated groups. Caustic esophageal burn was created by instilling 15% NaOH in the distalesophagus. The rats were left untreated or treated with 10 mg/kg per day MEG intraperitoneally for 5days. All rats were killed at 28 days. Efficacy of the treatment was assessed both histopathologicallyand biochemically.Results: Of 15 rats, 6 (40%) died in the untreated group, and only 1 (7%) rat died in the treated group.The stenosis index (SI) and the histopathologic damage score were significantly lower in the MEGtreatment group than the untreated group, which showed a correlation with tissue hydroxyproline level.In the untreated group, tissue oxidative stress parameters (malondialdehyde and protein carbonylcontent) were significantly higher; and antioxidant enzyme activities (superoxide dismutase andglutathione peroxidase) were significantly lower. Administration of MEG ameliorated oxidative stressparameters and antioxidant enzyme activities. Urinary nitrate and nitrite levels increased in the treatedand untreated groups in the first 3 days, suggesting increased nitrosative stress; but at the fourth day,nitrate and nitrite level reached control values in the treated group.Conclusion: Peroxynitrites play an important role in the healing process of caustic esophagitis. As aperoxynitrites scavenger, MEG potentially might be a useful adjuvant agent in the treatment ofesophageal caustic burn by modulating the antioxidant defense mechanism.© 2011 Elsevier Inc. All rights reserved.

⁎ Corresponding author. Department of Pediatric Surgery, Gulhaneilitary Medical Faculty, 06017 Etlik, Ankara, Turkey. Tel.: +90 312045483; fax: +90 312 3042150.E-mail address: drahmetguven@yahoo.com (A. Guven).

022-3468/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.jpedsurg.2011.02.060

Corrosive ingestions in children have some peculiarfeatures, such as esophageal caustic strictures, requiringdilatation that are often difficult and carry a high recurrencerate [1-3]. Unfortunately, long-term dilatation programs are

1747MEG attenuates caustic esophageal injury in rats

very challenging and require repeated hospital visits, loss ofwork for parents, and neglect of other children at home [3].Corrosive ingestion is an ongoing hazard despite aggressiveaccident prevention programs aimed at children, adulteducation, preventive labeling and packaging, and evenlegislation, limiting the strength and availability of causticsubstances [1,4,5].

The optimal approach to minimize esophageal damageafter caustic ingestion is controversial. A wide variety oftreatment modalities have been suggested to prevent fibrosissuch as steroids, antibiotics, proton pump inhibitors, frequentdilatation, intraluminal stents, and esophageal replacement[4,6-8]. Because none of these clinical approaches gainedpopularity, several studies were conducted to both clarify thepathophysiology of esophageal burn and reduce the risk ofstricture formation [9-13]. In light of these studies, the mainpurpose of medical treatment is to modify the inflammatoryresponse both at the site of the burn and in the deeper tissue,with the ultimate goal of less extensive scarring so as toprevent stricture formation.

Several studies have shown that inflammation seen inthe injured esophageal wall is characterized by decreasedtissue perfusion and increased breakdown of cellularmembranes by lipid peroxidation and hydrolysis [13-18].Reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) are believed to have a significant role inthe postischemic tissue damage that cause cellular injuryand subsequent necrosis via different mechanisms. Asseen in thermal injuries of the skin [18], free oxygenradicals play an important role in the acute stage of anesophageal burn [14-16,19], which correlated with ourprevious studies [13,20]. After the acute phase, scarformation begins when fibroblast proliferation replaces thesubmucosa and muscularis with commencement ofstricture formation. The end result may be a fibrotic andshortened esophagus [2,4,21].

Mercaptoethylguanidine (MEG) and related mercaptoalk-ylguanidines are potent scavengers of peroxynitrite andinhibit multiple peroxynitrite-induced oxidative processes[22,23]. Mercaptoethylguanidine has a dramatic protectiveeffect in many experimental models of inflammationincluding ischemia/reperfusion injury [23], periodontitis[24], hemorrhagic shock [25], inflammatory bowel disease[26], and endotoxic and septic shock [25].

In this experiment, our aim was to clarify the role forscavenging peroxynitrite in caustic esophageal injury. Wealso hypothesized that scavenging of peroxynitrite withMEG may prevent the development of fibrosis after causticesophageal injury in rats by modulating nitrosative stress.

1. Materials and methods

This study was approved by the local Ethical Committeefor Animal Experimentation.

1.1. Study groups

Forty-five Sprague-Dawley rats weighing 200 to 250 gwere randomly allocated into 3 main groups, each with 15animals. The esophagus was left uninjured and treated with0.09% NaCl (1 mL/kg, intraperitoneal [i.p.]) in the sham-operated group. In the control group, the esophagus wasinjured and treated with 0.09% NaCl (1 mL/kg, i.p.). In thetreated group, the esophagus was injured and treated withMEG (10 mg/kg, i.p.) (Sigma-Aldrich Inc, Saint Louis, MO).Treatments were started 1 hour after the burn injury andcontinued for 5 consecutive days.

1.2. Surgery

The animals were kept in metabolic cage to collect theirurine for 24 hours before surgery and were fasted for 12hours before the procedures. Each rat was anesthetized usingxylasine hydrochloride (15 mg/kg) and ketamine hydrochlo-ride (100 mg/kg), i.p. The method described by Gehanno andGuedon [12] was used to create standard caustic esophagealburns. Using sterile surgical techniques, a midline laparot-omy was made; and 2 cm of the abdominal esophagus wasisolated and tied with 2/0 silk sutures distally and proximally.A 24F catheter cannula was placed into the isolated segmentthrough a gastric puncture. The esophageal injury wascreated by instilling 15% NaOH solution for 90 seconds untilslight translucency of the esophageal wall and branching ofthe vessels were noted, and then the solution was aspirated.Subsequently, distilled water was used to irrigate the injuredsegment for a 60-second period. In the sham-operated group,distal esophageal segments were instilled with 0.09% NaClsolution only. The laparotomy incision was closed, and 10mL of saline was administered subcutaneously in eachanimal. Rats were allowed to feed ad libitum from 24 hourspostoperatively and were killed on the 28th day. The ratswere kept in a metabolic cage for 5 days and then kept inidentical cages.

1.3. Sample collection

After surgery, all rats were kept in a metabolic cage for 5days after surgery; and urine samples were collected andfrozen at −80°C until assayed.

At the end of experimental period, all animals were killedwith decapitation without prior anesthetization; and the distal2-cm esophageal segments were harvested for biochemicaland histological evaluation. The euthanasia technique waschosen to harvest tissue chemically uncontaminated. It is alsoknown that ketamine given in an anesthetic dose suppressesproinflammatory cytokines and interferes with ROS/RNS[27]. Proximal portions of injured segments were placed in10% buffered formaldehyde solution. The distal portion ofthe transected abdominal esophagus was stored at −80°Cuntil assayed.

Table 1 Comparison of changes in the weight, SI, and histopathological damage score of groups

Weights SI Histopathologicdamage score0 days 28 days

Sham-operated group (n = 15) 231 ± 25 258 ± 23 0.4 ± 0.06 0Untreated group (n = 9) 245 ± 34 185 ± 41 a 1.4 ± 0.18 a 3.4 ± 0.6 a

Treated group (n = 14) 235 ± 56 232 ± 43 0.9 ± 0.08 b 1.5 ± 0.7 b

a Statistically significant from the sham-operated and treatment groups.b Statistically significant from the sham-operated group.

1748 A. Guven et al.

1.4. Histopathologic evaluation

Histopathologic analysis was performed in a blind manner.Segments for histological analysis were fixed in formalin, androutine procedures were performed. Paraffin sections werestained with hematoxylin and eosin and Masson trichrome formicroscopic evaluation. The esophageal wall thickness and thelumen diameter were measured to calculate the SI by imageanalysis system: SI = wall thickness/lumen diameter. Inaddition, tissue damage was scored on a scale in 3 differentcategories: collagen deposition in the submucosa; damage tothemuscularismucosa; and damage and collagen deposition inthe tunica muscularis, for a total score of 0 to 5 as described inour previous works [13,20].

1.5. Biochemical evaluation

The frozen tissues were homogenized in phosphate buffer(pH 7.4) by means of a homogenizer (Heidolph Diax 900;

Fig. 1 Representative sample of esophageal sections from the differeEsophageal section of a rat in the untreated group that shows marked hcontent, damage to the muscularis mucosa, and a marked increase in tunicwith hypertrophied muscle). C, Esophageal section from an animal in thincrease in submucosal collagen content, and an almost normal tunica mu

Heidolph Elektro GmbH, Kelhaim, Germany) on an ice cube.The supernatant was allocated into 2 to 3 in separate tubes andstored at −70°C. The protein content of tissue homogenateswas measured by the method of Lowry et al [28], with bovineserum albumin as the standard. Efficacy of treatment wasassessed by the tissue level of malondialdehyde (MDA) usingthe method of Ohkawa et al [29]; protein carbonyl content(PCC), using the method of Levine et al [30]; superoxidedismutase (SOD), using the method of Sun et al [31]; andglutathione peroxidase (GSH-Px), using the method of Pagliaand Valentine [32]. The collagen content of the esophaguswas determined by tissue hydroxyproline (HP) levels usingthe spectrometric method of Reddy and Enwemeka [33].

1.6. Determination of nitric oxide metabolites(nitrate and nitrite in urine)

Samples were assayed for nitrate and nitrite (NO2) (NOx)levels using a nitric oxide (NO) Colorimetric Assay Kit

nt groups. A, Esophageal section of the sham-operated group. B,ypertrophic mucosa, a significant increase in submucosal collagena muscularis collagen content. (Note the thickened esophageal walle treated group reveals a slightly hypertrophic mucosa, a minimalscularis. (Masson trichrome staining; original magnification, ×10.)

Table 2 Comparison of Biochemical Evaluation

Hydroxyproline (μg/mg tissue) SOD (U/mg protein) GSH-Px (U/mg protein) MDA (μmol/g protein)

Sham-operated group (n = 15) 2.43 ± 0.42 2095 ± 136 7.82 ± 1.8 2.84 ± 0.47Untreated group (n = 9) 6.74 ± 0.93 a 1540 ± 171 a 5.45 ± 0.9 a 5.14 ± 0.52 a

Treated group (n = 14) 4.35 ± 0.86 b 1847 ± 154 b 6.91 ± 1.2 b 3.89 ± 0.34a Statistically significant from the sham-operated and treatment groups.b Statistically significant from the sham-operated group.

1749MEG attenuates caustic esophageal injury in rats

(Merck KGaA, Darmstadt, Germany) according to themanufacturer's instructions, and the estimated amountswere expressed as μM.

1.7. Statistical analysis

All statistical analyses were carried out using SPSSstatistical software (SPSS for Windows, version 11.0; SPSS,Chicago, IL). All data were presented as mean ± SD.Differences in measured parameters among the 3 groupswere analyzed by the Kruskal-Wallis test. Dual comparisonsbetween groups that present significant values wereevaluated with the Mann-Whitney U test. Statisticalsignificance was accepted with a value of P b .05.

Fig. 2 Determination of NO metabolites (NOx in urine) from thecontrol, untreated, and treated groups of rats. Each point is the mean± SEM for groups. ⁎ indicates statistically significant from sham-operated group; x, statistically significant from sham-operated andtreated groups.

2. Results

Thirty-eight rats survived throughout the study. Although6 rats (40%) died in the untreated group, only 1 rat (7%) diedin the treatment group; all sham-operated rats survivedduring the study. The mortality rates were significantlyhigher in the untreated group compared with those of thetreatment and sham groups (P b .01). During the exper-imental period, the initial and final weights of the rats areshown in Table 1. Untreated rats lost more weight thantreated and sham-operated rats, and this difference wasstatistically significant (P b .05).

There was an increase in wall thickness and a decrease inlumen diameter of esophagi in the untreated group.Therefore, the SI was found significantly higher in theuntreated group than that of the treated and sham-operatedgroups (P b .05). (Table 1). There were significant differencesbetween groups based on damage score (Table 1). Mansontricrome staining in the untreated groups showed intensifiedcollagen accumulation in the submucosa and tunica muscu-laris layers of the esophagus wall. Esophageal section of ratstreated with MEG reveals slightly increased submucosal andtunica muscularis collagen content and well-preservedmuscle (Fig. 1). Mean damage scores were significantlyhigher in the untreated group when compared with the othergroups (P b .05).

Table 2 summarizes the biochemical findings. The HPlevels were significantly higher in the untreated group

compared with the treated and sham-operated rats (untreatedvs the others; P b .05), suggesting that collagen productionwas higher in the untreated group. Superoxide dismutaseand GSH-Px enzyme activities in the untreated group wassignificantly lower than the treated and sham-operatedgroups (untreated vs the others; P b .05). Administration ofMEG caused an increase in SOD and GSH-Px activity(treated vs untreated; P b .05) but still lower than the sham-operated group. As an oxidative stress parameter, MDA andPCC levels were significantly increased in the untreatedrats; there was a slight increase in the treatment groupcompared with the sham-operated group (untreated vs theothers; P b .05).

Determination of NO metabolites (NOx in the urine ofrats) is seen in Fig. 2. Urinary NOx excretion increasedalmost 4-fold in the untreated group after surgery at the firstday and stayed near this level for 4 consecutive days. Theseoutcomes provide evidence that nitrosative stress occurredwith caustic esophageal burn. Mercaptoethylguanidineadministration reduced NOx levels, and this decrement wasstatistically significant from the first postoperative day(treated vs untreated group; P b .05). After the third day,NOx level reached near control values in the treated group(treated vs sham operated; P N .05).

1750 A. Guven et al.

3. Discussion

This study showed that caustic esophageal burn promotesoxidative and nitrosative stress–associated cellular damage.We used MEG as a potent scavenger of peroxynitrite anddemonstrated that MEG attenuated the degree of lipidperoxidation, protein oxidation, and peroxynitrites leveland ameliorated the decrease of antioxidant enzymesactivities in the esophagus of rats subjected to caustic burninjury. In addition, the esophagus sections of rats treated withMEG showed a more normal morphology than those ofuntreated rats that correlated to the tissue HP levels.

Esophageal stricture caused by ingestion of corrosivematerials is a major clinical problem associated withsignificant morbidity. During the early stage of injury afteringestion of caustic materials, ischemia induces irreversiblecellular changes by lipid peroxidation and hydrolysis[13,16,34]. Studies with more rigorous investigation revealedthat oxidative stress has a significant role in the affected siteof the esophageal wall [14,15]. In addition, studies displayedthat a variety of antioxidant agents may help to preventstricture formation in a caustic esophageal burn model in rats[19,35-37]. The development of dense scar tissue at the injurysite is a later consequence of caustic esophageal injury [3,6].Therefore, it is crucial to try to find some way to control thevery early damage triggered by the caustic agent.

We found that oxidative stress markers (MDA and PCC)were increased and antioxidant enzyme activities (SOD andGSH-Px) were decreased in rats' esophagi subjected to anexperimental caustic burn injury. Furthermore, administra-tion of MEG significantly decreased tissue MDA and PCClevels (indices of tissue damage) and increased SOD andGSH-Px levels in the treated group. The accumulation ofROS/RNS and reduction in antioxidant enzyme activitieslead to damage in cellular components such as lipids andproteins. Malondialdehyde, one of the final products of lipidperoxidation, is frequently used to confirm the involvementof free radicals in cellular damage. Besides lipid peroxida-tion, the measurement of proteic damage by PCC content andantioxidant enzyme activities can be performed to quantifyROS/RNS. Presumably, caustic materials cause decreasedtissue perfusion; and this initial stress triggers the release ofproinflammatory and inflammatory mediators and migrationof activated polymorphonuclear leukocytes resulting inexcess generation of ROS and RNS [13,16,34]. To eliminatetoxic ROS/RNS, cells are equipped with various antioxidantdefense systems such as SOD, which converts O2 to H2O2,and GSH-Px, which breaks down H2O2 to H2O and O2,enzymes and antioxidant systems that protect the cell againstlipid peroxidation and protein oxidation. Our previous worksand other studies with more rigorous investigation revealedthat oxidative stress has a significant role in the affected siteof the injured esophageal wall [13-15,20]. A variety ofantioxidant agents may help to prevent stricture formation ina caustic esophageal burn model in rats [19,35-37]. Theseobservations strongly suggest that caustic esophageal injury

is associated with decreased antioxidant activity resulting inincreased tissue damage.

Nitric oxide synthesized by NO synthase (NOS) is one ofthe most important mediators in the pathophysiologicalchanges in tissues. Under physiologic conditions, NOmaintains vascular tone and inhibits aggregation andadhesion of neutrophils and platelets to vascular endotheli-um; these are beneficial aspects of NO function [15,23]. Lowlevels of NO production protect an organ in the early stagesof injury, whereas elevated and prolonged NO production byiNOS during the later stages of the insult result in orpotentiate organ injury. Peroxynitrite anion, a potent oxidantmolecule, is produced by the rapid reaction of NO andsuperoxide, a process that occurs almost instantaneously andcauses the nitration of cellular proteins with subsequent lossof protein structure and function. Therefore, it is presumedthat scavenging peroxynitrite at an early stage of inflamma-tion would prevent inflammation and subsequent tissuedamage in an esophageal caustic burn injury. To clarify thishypothesis, we administered MEG to rats subjected tocaustic esophageal burn injury.

We found that caustic esophageal burn caused asignificant increase in urine NOx and that MEG significantlydecreased the urine NOx level, suggesting reduced productionof peroxynitrite. Peroxynitrite is eventually converted to NO2

and/or nitrate, that is, NOx and excreted in the urine [23].Therefore, urinary NOx levels are used as an indirect butreliable indicator for peroxynitrite formation in vivo. In thisstudy, the beneficial effect of MEG observed could be theresult of the reaction of the sulfhydryl group of MEG withperoxynitrite via a direct reaction [22]. Peroxynitrite anion israpidly converted to peroxynitrous acid, and peroxynitrousacid will also undergo a rate-limiting transition to an activatedintermediate that can oxidize target molecules such as proteinand lipid resulting in inhibition of mitochondrial enzymes,lipid peroxidation, protein oxidation, DNA injury, and other.Inhibition of this process by thiol-containing compoundssuch as MEG is another possible explanation for preventionof cellular death and preservation of normal esophagealmorphology [22,38]. Guanidines (of which MEG is one)could also react with NO2, which may also explain thedecreased urine NOx level [38,39]. We believe thatperoxynitrite have a crucial role in caustic injury and suggestthat peroxynitrite scavenging (neutralization of peroxynitrite-triggered oxidative processes) may be a therapeuticallyimportant pharmacological property of caustic esophagealburn injury.

This study also displayed moderate collagen deposition inthe submucosa and damage to the muscularis mucosa andtunica muscularis in the treated group. These histopathologicfindings correlated to HP levels, suggesting a lesser degree ofcollagen production. Hydroxyproline is uniquely present incollagen and can be used to determine the amount of collagenpresent in various tissues [33]. Scavenging of peroxynitritewith MEG might improve wound healing in the esophagealwall at an early phase and prevent stricture formation.

1751MEG attenuates caustic esophageal injury in rats

The starting time of medication in this study was chosento reflect clinical scenario because children who ingestcaustic material are usually brought to emergency depart-ment in a few hours. However, further studies are needed toinvestigate the effect of MEG on late admission.

Although MEG is known to act as a potent scavenger ofperoxynitrite, it is also a novel anti-inflammatory compoundwith a combined mode of action including inhibition ofiNOS in vitro and in vivo [22,23,40] and a modest directinhibition of cyclooxygenase activity [22]. Based on thesedata, although our results demonstrate that MEG protectedthe esophagus against caustic burn injury by reducingproduction and/or scavenging of peroxynitrite, comparablestudies with antioxidants and selective iNOS inhibitors areneeded to reveal the pathophysiology of caustic esophagealburn injury. Whatever the basis for beneficial action of MEGmay be, our findings provide a rationale for consideringscavenging peroxynitrite to ameliorate stricture formationresulting from caustic esophageal burn in humans. Wesuggest that MEG may possibly reduce stricture formation atan early stage after caustic esophageal burn and, by doing so,may ultimately decrease the need for stricture dilatation.Based on these observations, efforts to develop a safe clinicaltherapeutic protocol for the use of agents to prevent fibrosisand subsequent chronic stricture seem in order.

Our results demonstrate that peroxynitrite plays animportant role in the healing process of caustic esophagitisin a rat model. Mercaptoethylguanidine might prove to be auseful adjuvant agent in the treatment of esophageal causticburn by modulating the antioxidant defense mechanism.

References

[1] Atabek C, Surer I, Demirbag S, et al. Increasing tendency incaustic esophageal burns and long-term polytetrafluorethylenestenting in severe cases: 10 years experience. J Pediatr Surg2007;42:636-40.

[2] de Jong AL, Macdonald R, Ein S, et al. Corrosive esophagitis inchildren: a 30-year review. Int J Pediatr Otorhinolaryngol 2001;57:203-11.

[3] Contini S, Swarray-Deen A, Scarpignato C. Oesophageal corrosiveinjuries in children: a forgotten social and health challenge indeveloping countries. Bull World Health Organ 2009;87:950-4.

[4] Keh SM, Onyekwelu N, McManus K, et al. Corrosive injury to uppergastrointestinal tract: still a major surgical dilemma. World JGastroenterol 2006;12:5223-8.

[5] Guven A. Dangers to children at home: corrosive esophageal burn.TAF Prev Med Bull 2008;7:535-40.

[6] Karnak I, Tanyel FC, Buyukpamukcu N, et al. Combined use ofsteroid, antibiotics and early bougienage against stricture formationfollowing caustic esophageal burns. J Cardiovasc Surg (Torino)1999;40:307-10.

[7] Mutaf O. Treatment of corrosive esophageal strictures by long-termstenting. J Pediatr Surg 1996;31:681-5.

[8] Mutaf O, Ozok G, Avanoglu A. Oesophagoplasty in the treatment ofcaustic oesophageal strictures in children. Br J Surg 1995;82:644-6.

[9] Bautista A, Tojo R, Varela R, et al. Effects of prednisolone anddexamethasone on alkali burns of the esophagus in rabbit. J PediatrGastroenterol Nutr 1996;22:275-83.

[10] Bingol-Kologlu M, Tanyel FC, Muftuoglu S, et al. The preventiveeffect of heparin on stricture formation after caustic esophageal burns.J Pediatr Surg 1999;34:291-4.

[11] Demirbilek S, Bernay F, Rizalar R, et al. Effects of estradiol andprogesterone on the synthesis of collagen in corrosive esophagealburns in rats. J Pediatr Surg 1994;29:1425-8.

[12] Gehanno P, Guedon C. Inhibition of experimental esophageal lyestrictures by penicillamine. Arch Otolaryngol 1981;107:145-7.

[13] Guven A, Gundogdu G, Sadir S, et al. The efficacy of ozone therapy inexperimental caustic esophageal burn. J Pediatr Surg 2008;43:1679-84.

[14] Gunel E, Caglayan F, Caglayan O, et al. Reactive oxygen radical levelsin caustic esophageal burns. J Pediatr Surg 1999;34:405-7.

[15] Ozel SK, Dagli TE, Yuksel M, et al. The roles of free oxygen radicals,nitric oxide, and endothelin in caustic injury of rat esophagus. J PediatrSurg 2004;39:1381-5.

[16] Gunel E, Caglayan F, Caglayan O, et al. Effect of antioxidant therapyon collagen synthesis in corrosive esophageal burns. Pediatr Surg Int2002;18:24-7.

[17] Haller Jr JA, Andrews HG, White JJ, et al. Pathophysiology andmanagement of acute corrosive burns of the esophagus: results oftreatment in 285 children. J Pediatr Surg 1971;6:578-84.

[18] Horton JW. Free radicals and lipid peroxidation mediated injury in burntrauma: the role of antioxidant therapy. Toxicology 2003;189:75-88.

[19] Ocakci A, Coskun O, Tumkaya L, et al. Beneficial effects of Ebselenon corrosive esophageal burns of rats. Int J Pediatr Otorhinolaryngol2006;70:45-52.

[20] Guven A, Demirbag S, Uysal B, et al. Effect of 3-amino benzamide, apoly(adenosine diphosphate-ribose) polymerase inhibitor, in experi-mental caustic esophageal burn. J Pediatr Surg 2008;43:1474-9.

[21] Isolauri J, Reinikainen P, Markkula H. Functional evaluation ofinterposed colon in esophagus. Manometric and 24-hour pH observa-tions. Acta Chir Scand 1987;153:21-4.

[22] Szabo C, Ferrer-Sueta G, Zingarelli B, et al. Mercaptoethylguanidineand guanidine inhibitors of nitric-oxide synthase react with peroxyni-trite and protect against peroxynitrite-induced oxidative damage. J BiolChem 1997;272:9030-6.

[23] Guven A, Uysal B, Akgul O, et al. Scavenging of peroxynitrite reducesrenal ischemia/reperfusion injury. Ren Fail 2008;30:747-54.

[24] Lohinai Z, Benedek P, Feher E, et al. Protective effects ofmercaptoethylguanidine, a selective inhibitor of inducible nitricoxide synthase, in ligature-induced periodontitis in the rat. Br JPharmacol 1998;123:353-60.

[25] Szabo A, Hake P, Salzman AL, et al. Beneficial effects ofmercaptoethylguanidine, an inhibitor of the inducible isoform of nitricoxide synthase and a scavenger of peroxynitrite, in a porcine model ofdelayed hemorrhagic shock. Crit Care Med 1999;27:1343-50.

[26] Zingarelli B, Cuzzocrea S, Szabo C, et al. Mercaptoethylguanidine, acombined inhibitor of nitric oxide synthase and peroxynitritescavenger, reduces trinitrobenzene sulfonic acid–induced colonicdamage in rats. J Pharmacol Exp Ther 1998;287:1048-55.

[27] Ergun Y, Darendeli S, Imrek S, et al. The comparison of the effects ofanesthetic doses of ketamine, propofol, and etomidate on ischemia-reperfusion injury in skeletal muscle. Fundam Clin Pharmacol2010;24:215-22.

[28] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement withthe Folin phenol reagent. J Biol Chem 1951;193:265-75.

[29] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animaltissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8.

[30] Levine RL, Garland D, Oliver CN, et al. Determination of carbonylcontent in oxidatively modified proteins. Methods Enzymol 1990;186:464-78.

[31] Sun Y, Oberley LW, Li Y. A simple method for clinical assay ofsuperoxide dismutase. Clin Chem 1988;34:497-500.

[32] Paglia DE, Valentine WN. Studies on the quantitative and qualitativecharacterization of erythrocyte glutathione peroxidase. J Lab Clin Med1967;70:158-69.

1752 A. Guven et al.

[33] Reddy GK, Enwemeka CS. A simplified method for the analysisof hydroxyproline in biological tissues. Clin Biochem 1996;29:225-9.

[34] Demling RH, Lalonde C. Systemic lipid peroxidation and inflamma-tion induced by thermal injury persists into the post-resuscitationperiod. J Trauma 1990;30:69-74.

[35] Basaran UN, Eskiocak S, Altaner S, et al. Inhibition of iNOS with S-methylisothiourea was impaired in wound healing in causticesophageal burn. Int J Pediatr Otorhinolaryngol 2005;69:471-7.

[36] Larios-Arceo F, Ortiz GG, Huerta M, et al. Protective effects ofmelatonin against caustic esophageal burn injury in rats. J Pineal Res2008;45:219-23.

[37] Liu AJ, Richardson MA. Effects of N-acetylcysteine on experimen-tally induced esophageal lye injury. Ann Otol Rhinol Laryngol1985;94:477-82.

[38] Quijano C, Alvarez B, Gatti RM, et al. Pathways of peroxynitriteoxidation of thiol groups. Biochem J 1997;322(Pt 1):167-73.

[39] van der Vliet A, Eiserich JP, O'Neill CA, et al. Tyrosine modificationby reactive nitrogen species: a closer look. Arch Biochem Biophys1995;319:341-9.

[40] Southan GJ, Zingarelli B, O'Connor M, et al. Spontaneousrearrangement of aminoalkylisothioureas into mercaptoalkylguani-dines, a novel class of nitric oxide synthase inhibitors with selectivitytowards the inducible isoform. Br J Pharmacol 1996;117:619-32.