www.elsevier.com/locate/jpedsurg

Journal of Pediatric Surgery (2008) 43, 1474–1479

Effect of 3-amino benzamide, a poly(adenosinediphosphate-ribose) polymerase inhibitor, inexperimental caustic esophageal burnAhmet Guvena,⁎, Suzi Demirbaga, Bulent Uysalb, Turgut Topalb, Esra Erdoganc,Ahmet Korkmazb, Haluk Ozturka

aDepartment of Pediatric Surgery, Gulhane Military Medical Academy, Etlik, 06017 Ankara, TurkeybDepartment of Physiology, Gulhane Military Medical Academy, Etlik, 06017 Ankara, TurkeycDepartment of Histology and Embryology, Gulhane Military Medical Academy, Etlik, 06017 Ankara, Turkey

Received 11 August 2007; revised 3 October 2007; accepted 3 October 2007

0d

Key words:Caustic esophageal burn;3-Amino benzamide;Poly(ADP-ribose)polymerase inhibition;

Antioxidant enzymes;Lipid peroxidation;Protein oxidation

AbstractIntroduction: The enzyme poly(adenosine diphosphate-ribose) polymerase affects the repair of DNA indamaged cells. However, its activation in damaged cells can lead to adenosine triphosphate depletionand death. This study was designed to investigate the efficacy of 3-amino benzamide (3-AB), a poly(adenosine diphosphate-ribose) polymerase inhibitor, on the prevention of esophageal damage andstricture-formation development after esophageal caustic injuries in rat.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 to the distal esophagus.The rats were left untreated or treated with 3-AB 10 mg/kg per day intraperitoneally. All rats were killedon the 28th day. Efficacy of the treatment was assessed by measuring the stenosis index andhistopathologic damage score and biochemically by determining tissue hydroxyproline content,superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), and proteincarbonyl content (PCC) in esophageal homogenates.Results: Treatment with 3-AB decreased the stenosis index and histopathologic damage score seen incaustic esophageal burn rats. Hydroxyproline level was significantly higher in the untreated group ascompared with the group treated with 3-AB. Caustic esophageal burn increased MDA and PCC levelsand also decreased SOD and GPx enzyme activities. On the contrary, 3-AB treatment decreased theelevated MDA and PCC levels and also increased the reduced SOD and GPx enzyme activities.Conclusion: 3-Amino benzamide has a preventive effect in the development of fibrosis by decreasingtissue damage and increasing the antioxidant enzyme activity in an experimental model of corrosiveesophagitis in rats.© 2008 Elsevier Inc. All rights reserved.

⁎ Corresponding author. Tel.: +90 312 3045483; fax: +90 312 3042150.E-mail address: drahmetguven@yahoo.com (A. Guven).

022-3468/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.jpedsurg.2007.10.001

Caustic esophageal burn, which leads to strictureformation as a late complication, is a common problemin the pediatric age group [1,2]. After ingestion of caustic

1475Effect of 3-AB, a poly(adenosine diphosphate-ribose) polymerase inhibitor

material, inflammation characterized by decreased tissueperfusion and increased breakdown of cellular membranesby lipid peroxidation and hydrolysis is seen at the injurysite [3]. It is well known that postischemic tissue damageis caused by reactive oxygen species (ROS) and reactivenitrogen species. Gunel et al [4,5] had found that ROSplayed a role in the early phase of esophageal burn asseen in the thermal injuries of the skin [6]. After theimmediate phase, scar formation begins with fibroblasticproliferation and synthesis, deposition, and remodeling ofthe collagen.

Although many treatment modalities have been suggestedto prevent fibrosis, including steroids, frequent dilatation,intraluminal stents, and esophageal replacement [3,7,8], theoptimal management protocol to treat the damage afteringestion of caustic materials remains controversial. Becausenone of these clinical approaches gained popularity, severalresearches regarding vitamins C and E, heparin, caffeic acidphenethyl ester, epidermal growth factor, interferon γ,penicillamine, sphingosylphosphorylcholine, estradiol, andprogesterone were conducted to reduce the risk of strictureformation [9-15]. The main purpose of medical treatment isto inhibit inflammatory reaction at the early phase so as toprevent stricture formation.

There is evidence that inflammation (ROS/reactivenitrogen species) induces cellular injury by inducingnicks in DNA. Deoxyribonucleic acid damage is repairedvia the activity of several DNA repair enzymes, includingpoly(adenosine diphosphate-ribose) polymerase (PARP).Extensive DNA damage may lead to excessive PARPactivation that consumes large quantities of cellularnicotinamide adenine dinucleotide, resulting in adenosinetriphosphate (ATP) depletion and cellular death. Therefore,it has been hypothesized that inhibition of activity ofPARP may prevent cell death via increasing cellular ATPlevel. In this study, we focused on the preventive effect of3-amino benzamide (3-AB), PARP inhibitor, on lipidperoxidation, protein oxidation, antioxidant enzyme sys-tem, and development of fibrosis after caustic esophagealinjuries in rats.

1. Materials and methods

This study was approved by the experimental ethicscommittee of Gulhane Military Medical Academy,Ankara, Turkey.

1.1. Study groups

Forty-five Sprague-Dawley rats weighing 250 to 300 gwere randomly allocated into 3 main groups of 15 animals ineach. The esophagus was uninjured and untreated in thesham-operated group. In control group, the esophagus wasinjured and left untreated. In the treatment group, the

esophagus was injured and treated with intraperitoneal 3-AB(10 mg/kg) immediately after the burn injury and continuedfor 5 consecutive days.

1.2. Surgery

The animals were fed with standard raw chow andwater ad libitum and were fasted for 12 hours before theprocedures. Each rat was anesthetized using xylasinehydrochloride (15 mg/kg) and ketamine hydrochloride(100 mg/kg) intraperitoneally. The method described byGehanno and Guedon [12] was used to create standardcaustic esophageal burns. A midline laparotomy was made,using sterile surgical techniques, and 2 cm of abdominalesophageal segment was isolated and tied with 2/0 silksutures distally and proximally. A 24F cannula was placedinto the isolated segment through a gastric puncture. Theesophageal injury was created by instilling 15% NaOHsolution for 3 minutes under slight translucency of theesophagial wall, and branching of the vessels were noted;the solution was then aspirated. Subsequently, distilledwater was used to irrigate the burned area segment for a60-second period. In the sham-operated group, distalesophageal segments were instilled with 0.09% NaClsolution only. The laparotomy incision was closed, and10 mL of saline was administered subcutaneously in eachanimal. The animals were not allowed to feed for the next24 hours. All animals were kept in identical cages thatprovided food and water during the study period and werekilled on the 28th day.

1.3. Sample collection

At the end of experiment period, all animals were killedby decapitation, and distal 2-cm esophageal segments wereharvested for biochemical and histologic evaluation inves-tigation. Proximal portions of burned segments were placedinto 10% buffered formaldehyde solution. The distal portionof the transected abdominal esophagus was stored in −80°Cuntil assayed.

1.4. Histopathologic evaluation

Histopathologic analysis was performed in a blindmanner. Segments for histology were fixed in formalin,and routine procedures were performed. Paraffin sectionswere stained with hematoxylin-eosin and Masson trichromefor microscopic evaluation. The esophageal wall thicknessand the lumen diameter were measured to calculate thestenosis index (SI) by image analysis system: SI = [wallthickness]/[lumen diameter]. In addition, tissue damage wasscored on a scale in 3 different categories, collagendeposition in the submucosa, damage to the muscularismucosa, and damage and collagen deposition in the tunicamuscularis, for a total score of 0 to 5 (Table 1).

Table 1 Criteria for histopathologic evaluation

Criterion score

Increase in submucosal collagenNone 0Mild (submucosal collagen at least twice the thicknessof the muscularis mucosa)

1

Marked (submucosal collagen more than twice thethickness of the muscularis mucosa)

2

Damage to the muscularis mucosaNone 0Present 1Damage and collagen deposition in thetunica muscularisNone 0Mild (collagen deposition around the smoothmuscle fibers)

1

Marked (same as mild, with collagen depositionreplacing some of the fibers)

2

1476 A. Guven et al.

1.5. Biochemical evaluation

The frozen tissues were homogenized in phosphate buffer(pH 7.4) by means of a homogenizator (Heidolph Diax 900;Heidolph Elektro GmbH, Kelhaim, Germany) in an ice cube.The supernatant was allocated into 2 to 3 in separate tubesand stored at −70°C again. First of all, the protein content oftissue homogenates was measured by the method of Lowryet al [16] with bovine serum albumin as the standard.Efficacy of treatment was assessed by tissue level of malon-dialdehyde (MDA) using the method of Ohkawa et al [17],protein carbonyl content (PCC) using the method of Levineet al [18], superoxide dismutase (SOD) using the method ofSun et al [19], and glutathione peroxidase (GPx) using themethod of Paglia and Valentine [20]. Collagen content ofesophagus was determined by tissue hydroxyproline (HP)levels using the spectrometric method of Reddy andEnwemeka [21].

1.6. Statistical analysis

All statistical analyses were carried out using SPSSstatistical software (SPSS for Windows, Version 11.0, SPSS,Chicago, Ill). All data were presented as mean ± SD.

Table 2 Comparison of changes in the weight, SI, and histopatholog

Weight

Zeroth Day 2

Sham-operated group (n = 15) 326 ± 12 3Untreated group (n = 9) 335 ± 15 2Treatment group (n = 13) 329 ± 13 3

⁎ Statistically significant from sham-operated and treatment group.† Statistically significant from sham-operated group.

Differences in measured parameters among the 3 groupswere analyzed by Kruskal-Wallis test. Dual comparisonsbetween groups that present significant values wereevaluated with Mann-Whitney U test. Statistical significancewas accepted at P b .05.

2. Results

Thirty-seven rats survived throughout the study. Six(40%) rats in the untreated group died, only 2 (13%) rats inthe treated group died during the study, and all sham ratssurvived. The mortality rates were significantly higher in theuntreated group compared with treated and sham groups (P b.05). There was no statistically significant difference betweenthe treated and sham-operated groups. During the killing,esophagi of the rats in treated group were macroscopicallynormal except slight adhesions. On the contrary, those ofuntreated animals showed considerable complete esophagealobstruction, severe adhesion (possibly because of perfora-tion), and feed residue in the esophageal lumen. All animalssurvived, and there was no complication in the sham-operated group.

The initial and eventual weights of rats during theexperiment period are shown in Table 2. There weresignificant differences at the end of experiment periodbetween untreated and treated rats, and also, difference wassignificant between the treated and sham groups (P b .05).

The SI and histopathologic grading scores are shown inTable 2. The SI in the untreated group was significantlyhigher than in the sham-operated and 3-AB treatment groups(P b .05). There were significant differences between groupsbased on damage score (Fig. 1). Collagen accumulation inthe submucosa and tunica muscularis was significantlyhigher in the untreated group when compared with thetreated group (P b .05). In 3-AB treatment group, there was amild increase in the submucosal and tunica musculariscollagen accumulation when compared with the sham group.

The biochemical findings are shown in Table 3. The HPlevels were significantly higher in the untreated groupcompared with the treated and sham groups (P b .05), and themean production of HP was significantly higher in treatedgroup than in the sham group (P b .05). To assess the effect ofoxidative stress, SOD and GPx enzyme activities were

ic damage score of groups

SI Histopathologicdamage score8th Day

52 ± 13 0.4 ± 0.04 064 ± 35 ⁎ 1.2 ± 0.08 ⁎ 3.5 ± 0.2 ⁎

14 ± 32 † 0.6 ± 0.05 † 1.4 ± 0.2 †

Fig. 1 Representative sample of the esophageal section of the groups. A, Esophageal section of the sham-operated group. B, Esophagealsection of the untreated group reveals marked hypertrophic mucosa, significant increase in submucosal collagen content, damage to muscularismucosa, and marked increase in tunica muscularis collagen content. (Note the thickened esophageal wall.) C, Esophageal section of the grouptreated with 3-AB reveals slightly hypertrophic mucosa, minimal increase in submucosal collagen content, and almost normal tunicamuscularis (Masson trichrome staining, original magnification ×10).

1477Effect of 3-AB, a poly(adenosine diphosphate-ribose) polymerase inhibitor

evaluated. Superoxide dismutase and GPx enzyme activitieswere significantly lower in the untreated group (P b .05),whereas enzyme activities in the treated group were similarto sham group. Although MDA and PCC were significantlyincreased in the untreated group, there was a slight increasein the treated group compared with the sham group.

3. Discussion

In this caustic esophageal burn model, we used 3-AB asPARP inhibitor that has not been tried before in such amodel. We demonstrated that 3-AB administration attenu-ated the elevation of MDA and PCC levels and amelioratedthe decrease of SOD and GPx activity. It is of interest that at28 days postburned, esophagus sections from 3-AB–treatedrats appear more normal than those of untreated rats and alsoconfirmed this protective effect of 3-AB.

Esophageal strictures caused by ingestion of corrosivematerials are a major clinical problem with a high degreeof morbidity. Most clinicians still use the tedious practiceof recurrent esophageal dilatations, facing the problem ofrecurrent fibrosis and stricture after the dilatation.

Several lines of evidence indicate that ROS are generatedat the burn site immediately after injury [4-6,22]. Gunel et al

Table 3 Comparison of biochemical evaluations

HP(μg/mg tissue)

SOD(U/mg protein)

Sham-operated group(n = 15)

1.4 ± 0.2 21.3 ± 1.2

Untreated group (n = 9) 3.1 ± 0.6 ⁎ 10.4 ± 1.0 ⁎

Treatment group (n = 13) 1.7 ± 0.3 20.1 ± 1.7

⁎ Statistically significant from sham-operated and treatment group.† Statistically significant from sham-operated group.

[4,5] have shown that tissue oxygen radical levels aresignificantly increased and antioxidant capacity is decreased72 hours after caustic injury. And it is well established thatcellular DNA damage results from the generation ofreactive radicals.

The regenerative capacity of tissue immediately afterinjury is dependent on the number of noninjured orsublethally injured cells that survive and can initiate thereparative mechanisms that restore the structure of the tissue[23]. One of the major requirements for initiation of thecellular repair process is the repletion of intracellular ATP[6,24,25]. On the other hand, extensive DNA damage afterinjury because of generated oxygen radicals may lead toexcessive PARP activation that consumes large quantities ofcellular nicotinamide adenine dinucleotide resulting in ATPdepletion and dysfunction or necrotic cell death [24].Although we did not determine whether PARP inhibitionincreased levels of tissue ATP postexposure to NaOH, ourdata showed that PARP inhibition restores cell injuries. Thisis consistent with decreased level of MDA, PCC (indices oftissue damage), HP, and ameliorated histopathologic findingsin our work.

We found that tissue SOD and GPx enzyme activity weredecreased in untreated rats, whereas those were increased inrats that received 3-AB. Moreover, tissue MDA and PCClevels were found significantly increased in the untreated

GPx(U/mg protein)

MDA(μmol/g protein)

PCC(μmol/g protein)

33.2 ± 1.8 0.30 ± 0.02 0.39 ± 0.01

22.1 ± 1.8 ⁎ 0.55 ± 0.03 ⁎ 0.59 ± 0.02 ⁎

31.5 ± 2.8 0.35 ± 0.03 0.45 ± 0.02 †

1478 A. Guven et al.

group and decreased in the treated group. The antioxidantsystems protect the cell against lipid peroxidation andprotein oxidation. These results consistent with reactiveoxygen radicals play an important role in caustic esophagus.Gunel et al [4] reported that MDA level was increased, andglutation level (antioxidant substance that augments glu-tathione GPx activity) was decreased at 24 hours andremained high for 72 hours after caustic injury. However,they did not evaluate the results in late period. Ocakci et al[26] showed that MDA was increased, and SOD, GPx, andcatalase were decreased at 28 days in corrosive esophagitisin rats. We can conclude that caustic esophagitis isassociated with increased reactive oxygen radicals andtissue damage. In addition to this, 3-AB may be a possiblebeneficial agent to reduce tissue damage by enhancedantioxidant enzyme activity.

One could argue that the beneficial effects of 3-AB arebecause of its ability to scavenge free radicals. There isevidence that the PARP inhibitors scavenge reactive oxygenradicals. This notion is supported by our finding that PARPinhibitor was able to ameliorate the tissue SOD and GPxenzyme activity determined to evaluate antioxidant capacityof the tissue. To date, there has been no research into the roleof PARP within the esophagus under normal and pathophy-siologic conditions. However, several experimental studiesshowed that the inhibition of PARP-activation pathwayameliorates the tissue injury because of free radicals invarious forms of shock, inflammation, trauma, and reperfu-sion injury [23,24,27,28]. From this study, it can beconcluded that 3-AB have antioxidant effect in causticesophageal burn in rats.

In the present study, administration of 3-AB during thefirst 5 days after caustic injury significantly decreased the HPlevel, SI, and histopathologic injury score compared with theuntreated rat. Collagen deposition in the submucosa anddamage to the muscularis mucosa and tunica muscularis intreated group were less than that in untreated group. Thesehistopathologic findings showed correlation with biochem-ical results. It is well known that alkaline caustic agents aredestructive to the esophagus by a process of liquefactionnecrosis [3,9]. Therefore, preventing stricture formationshould be supporting normal wound healing in esophagealwall at early phase. It was seen that PARP-inhibitor treatmentsignificantly reduced neuronal [29], retinal [30], and heart orskeletal muscle [28]. Furthermore, 3-AB decreased progres-sion of postinfarction heart interstitial fibrosis [28,31]. Takentogether, it might be possible to prevent smooth muscle deathby prevention of ATP depletion in cells, which decreased thefibroplasia during healing of corrosive esophageal burns byusing 3-AB.

The starting time of medication in the studies related toPARP inhibitors is generally before the injury [24,27,30]. Incontrast to these studies, we started with treatment with 3-ABimmediately after the burn was created. After exposure,victims, especially children, can arrive to the emergencydepartment in a few hours. Because metabolic rate of

rodents, especially rats, is almost 10-fold faster than that ofhumans, to start medication in a few minutes after injuryseems to be realistic. Nevertheless, time-course studies arenecessary to determine the starting time after the injury.

It is also necessary to design further studies to clarifypathophysiologic mechanism of caustic burn and todetermine whether the beneficial effect of this agent isirreversible. Inhibition of PARP, especially chronic inhibi-tion, may be harmful because this enzyme facilitates DNArepair that may be required in the recovery of esophagus inlater phase. For this reason, determination of the propertiming for PARP inhibition postburned period may be acrucial factor to permit the use of PARP inhibitors in causticesophageal burn.

However, whatever the basis for beneficial action of 3-ABmay be, our findings provide a rationale for using ofinhibitors of PARP to ameliorate stricture formation becauseof caustic burn in humans.

In conclusion, our results strongly showed that PARPplays an important role in the healing process in causticesophagitis, and as a PARP inhibitor, 3-AB might be apotential agent to treat esophageal caustic burn byincreasing antioxidant defense mechanism, decreasing tissuedamage, and ameliorating histopathologic injuries. Furtherstudies should be assessed to determine the side effect ofthis agent.

Acknowledgment

We thank Dr Gokhan Gundogdu for the technicalassistance during the experimental procedures.

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