Journal of Pediatric Surgery (2008) 43, 1474–1479
www.elsevier.com/locate/jpedsurg
Effect of 3-amino benzamide, a poly(adenosine diphosphate-ribose) polymerase inhibitor, in experimental caustic esophageal burn Ahmet Guven a,⁎, Suzi Demirbag a , Bulent Uysal b , Turgut Topal b , Esra Erdogan c , Ahmet Korkmaz b , Haluk Ozturk a a
Department of Pediatric Surgery, Gulhane Military Medical Academy, Etlik, 06017 Ankara, Turkey Department of Physiology, Gulhane Military Medical Academy, Etlik, 06017 Ankara, Turkey c Department of Histology and Embryology, Gulhane Military Medical Academy, Etlik, 06017 Ankara, Turkey b
Received 11 August 2007; revised 3 October 2007; accepted 3 October 2007
Key words: Caustic esophageal burn; 3-Amino benzamide; Poly(ADP-ribose) polymerase inhibition; Antioxidant enzymes; Lipid peroxidation; Protein oxidation
Abstract Introduction: The enzyme poly(adenosine diphosphate-ribose) polymerase affects the repair of DNA in damaged cells. However, its activation in damaged cells can lead to adenosine triphosphate depletion and 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 and stricture-formation development after esophageal caustic injuries in rat. Materials and Methods: Forty-five rats were allocated into 3 groups: sham-operated, untreated, and treated 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 killed on the 28th day. Efficacy of the treatment was assessed by measuring the stenosis index and histopathologic damage score and biochemically by determining tissue hydroxyproline content, superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), and protein carbonyl content (PCC) in esophageal homogenates. Results: Treatment with 3-AB decreased the stenosis index and histopathologic damage score seen in caustic esophageal burn rats. Hydroxyproline level was significantly higher in the untreated group as compared with the group treated with 3-AB. Caustic esophageal burn increased MDA and PCC levels and also decreased SOD and GPx enzyme activities. On the contrary, 3-AB treatment decreased the elevated 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 decreasing tissue damage and increasing the antioxidant enzyme activity in an experimental model of corrosive esophagitis in rats. © 2008 Elsevier Inc. All rights reserved.
⁎ Corresponding author. Tel.: +90 312 3045483; fax: +90 312 3042150. E-mail address:
[email protected] (A. Guven). 0022-3468/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2007.10.001
Caustic esophageal burn, which leads to stricture formation as a late complication, is a common problem in the pediatric age group [1,2]. After ingestion of caustic
Effect of 3-AB, a poly(adenosine diphosphate-ribose) polymerase inhibitor material, inflammation characterized by decreased tissue perfusion and increased breakdown of cellular membranes by lipid peroxidation and hydrolysis is seen at the injury site [3]. It is well known that postischemic tissue damage is caused by reactive oxygen species (ROS) and reactive nitrogen species. Gunel et al [4,5] had found that ROS played a role in the early phase of esophageal burn as seen in the thermal injuries of the skin [6]. After the immediate phase, scar formation begins with fibroblastic proliferation and synthesis, deposition, and remodeling of the collagen. Although many treatment modalities have been suggested to prevent fibrosis, including steroids, frequent dilatation, intraluminal stents, and esophageal replacement [3,7,8], the optimal management protocol to treat the damage after ingestion of caustic materials remains controversial. Because none of these clinical approaches gained popularity, several researches regarding vitamins C and E, heparin, caffeic acid phenethyl ester, epidermal growth factor, interferon γ, penicillamine, sphingosylphosphorylcholine, estradiol, and progesterone were conducted to reduce the risk of stricture formation [9-15]. The main purpose of medical treatment is to inhibit inflammatory reaction at the early phase so as to prevent stricture formation. There is evidence that inflammation (ROS/reactive nitrogen species) induces cellular injury by inducing nicks in DNA. Deoxyribonucleic acid damage is repaired via the activity of several DNA repair enzymes, including poly(adenosine diphosphate-ribose) polymerase (PARP). Extensive DNA damage may lead to excessive PARP activation that consumes large quantities of cellular nicotinamide adenine dinucleotide, resulting in adenosine triphosphate (ATP) depletion and cellular death. Therefore, it has been hypothesized that inhibition of activity of PARP may prevent cell death via increasing cellular ATP level. In this study, we focused on the preventive effect of 3-amino benzamide (3-AB), PARP inhibitor, on lipid peroxidation, protein oxidation, antioxidant enzyme system, and development of fibrosis after caustic esophageal injuries in rats.
1. Materials and methods This study was approved by the experimental ethics committee of Gulhane Military Medical Academy, Ankara, Turkey.
1.1. Study groups Forty-five Sprague-Dawley rats weighing 250 to 300 g were randomly allocated into 3 main groups of 15 animals in each. The esophagus was uninjured and untreated in the sham-operated group. In control group, the esophagus was injured and left untreated. In the treatment group, the
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esophagus was injured and treated with intraperitoneal 3-AB (10 mg/kg) immediately after the burn injury and continued for 5 consecutive days.
1.2. Surgery The animals were fed with standard raw chow and water ad libitum and were fasted for 12 hours before the procedures. Each rat was anesthetized using xylasine hydrochloride (15 mg/kg) and ketamine hydrochloride (100 mg/kg) intraperitoneally. The method described by Gehanno and Guedon [12] was used to create standard caustic esophageal burns. A midline laparotomy was made, using sterile surgical techniques, and 2 cm of abdominal esophageal segment was isolated and tied with 2/0 silk sutures distally and proximally. A 24F cannula was placed into the isolated segment through a gastric puncture. The esophageal injury was created by instilling 15% NaOH solution for 3 minutes under slight translucency of the esophagial wall, and branching of the vessels were noted; the solution was then aspirated. Subsequently, distilled water was used to irrigate the burned area segment for a 60-second period. In the sham-operated group, distal esophageal segments were instilled with 0.09% NaCl solution only. The laparotomy incision was closed, and 10 mL of saline was administered subcutaneously in each animal. The animals were not allowed to feed for the next 24 hours. All animals were kept in identical cages that provided food and water during the study period and were killed on the 28th day.
1.3. Sample collection At the end of experiment period, all animals were killed by decapitation, and distal 2-cm esophageal segments were harvested for biochemical and histologic evaluation investigation. Proximal portions of burned segments were placed into 10% buffered formaldehyde solution. The distal portion of the transected abdominal esophagus was stored in −80°C until assayed.
1.4. Histopathologic evaluation Histopathologic analysis was performed in a blind manner. Segments for histology were fixed in formalin, and routine procedures were performed. Paraffin sections were stained with hematoxylin-eosin and Masson trichrome for microscopic evaluation. The esophageal wall thickness and the lumen diameter were measured to calculate the stenosis index (SI) by image analysis system: SI = [wall thickness]/[lumen diameter]. In addition, tissue damage was scored on a scale in 3 different categories, collagen deposition in the submucosa, damage to the muscularis mucosa, and damage and collagen deposition in the tunica muscularis, for a total score of 0 to 5 (Table 1).
1476 Table 1
A. Guven et al. Criteria for histopathologic evaluation
Criterion score Increase in submucosal collagen None Mild (submucosal collagen at least twice the thickness of the muscularis mucosa) Marked (submucosal collagen more than twice the thickness of the muscularis mucosa) Damage to the muscularis mucosa None Present Damage and collagen deposition in the tunica muscularis None Mild (collagen deposition around the smooth muscle fibers) Marked (same as mild, with collagen deposition replacing some of the fibers)
0 1 2
0 1
0 1 2
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 tubes and stored at −70°C again. First of all, the protein content of tissue homogenates was measured by the method of Lowry et al [16] with bovine serum albumin as the standard. Efficacy of treatment was assessed by tissue level of malondialdehyde (MDA) using the method of Ohkawa et al [17], protein carbonyl content (PCC) using the method of Levine et al [18], superoxide dismutase (SOD) using the method of Sun et al [19], and glutathione peroxidase (GPx) using the method of Paglia and Valentine [20]. Collagen content of esophagus was determined by tissue hydroxyproline (HP) levels using the spectrometric method of Reddy and Enwemeka [21].
1.6. Statistical analysis All statistical analyses were carried out using SPSS statistical software (SPSS for Windows, Version 11.0, SPSS, Chicago, Ill). All data were presented as mean ± SD.
Table 2
Differences in measured parameters among the 3 groups were analyzed by Kruskal-Wallis test. Dual comparisons between groups that present significant values were evaluated with Mann-Whitney U test. Statistical significance was 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 in the treated group died during the study, and all sham rats survived. The mortality rates were significantly higher in the untreated group compared with treated and sham groups (P b .05). There was no statistically significant difference between the treated and sham-operated groups. During the killing, esophagi of the rats in treated group were macroscopically normal except slight adhesions. On the contrary, those of untreated animals showed considerable complete esophageal obstruction, severe adhesion (possibly because of perforation), and feed residue in the esophageal lumen. All animals survived, and there was no complication in the shamoperated group. The initial and eventual weights of rats during the experiment period are shown in Table 2. There were significant differences at the end of experiment period between untreated and treated rats, and also, difference was significant between the treated and sham groups (P b .05). The SI and histopathologic grading scores are shown in Table 2. The SI in the untreated group was significantly higher than in the sham-operated and 3-AB treatment groups (P b .05). There were significant differences between groups based on damage score (Fig. 1). Collagen accumulation in the submucosa and tunica muscularis was significantly higher in the untreated group when compared with the treated group (P b .05). In 3-AB treatment group, there was a mild increase in the submucosal and tunica muscularis collagen accumulation when compared with the sham group. The biochemical findings are shown in Table 3. The HP levels were significantly higher in the untreated group compared with the treated and sham groups (P b .05), and the mean production of HP was significantly higher in treated group than in the sham group (P b .05). To assess the effect of oxidative stress, SOD and GPx enzyme activities were
Comparison of changes in the weight, SI, and histopathologic damage score of groups Weight
Sham-operated group (n = 15) Untreated group (n = 9) Treatment group (n = 13)
Zeroth Day
28th Day
326 ± 12 335 ± 15 329 ± 13
352 ± 13 264 ± 35 ⁎ 314 ± 32 †
⁎ Statistically significant from sham-operated and treatment group. † Statistically significant from sham-operated group.
SI
Histopathologic damage score
0.4 ± 0.04 1.2 ± 0.08 ⁎ 0.6 ± 0.05 †
0 3.5 ± 0.2 ⁎ 1.4 ± 0.2 †
Effect of 3-AB, a poly(adenosine diphosphate-ribose) polymerase inhibitor
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Fig. 1 Representative sample of the esophageal section of the groups. A, Esophageal section of the sham-operated group. B, Esophageal section of the untreated group reveals marked hypertrophic mucosa, significant increase in submucosal collagen content, damage to muscularis mucosa, and marked increase in tunica muscularis collagen content. (Note the thickened esophageal wall.) C, Esophageal section of the group treated with 3-AB reveals slightly hypertrophic mucosa, minimal increase in submucosal collagen content, and almost normal tunica muscularis (Masson trichrome staining, original magnification ×10).
evaluated. Superoxide dismutase and GPx enzyme activities were significantly lower in the untreated group (P b .05), whereas enzyme activities in the treated group were similar to sham group. Although MDA and PCC were significantly increased in the untreated group, there was a slight increase in the treated group compared with the sham group.
3. Discussion In this caustic esophageal burn model, we used 3-AB as PARP inhibitor that has not been tried before in such a model. We demonstrated that 3-AB administration attenuated the elevation of MDA and PCC levels and ameliorated the decrease of SOD and GPx activity. It is of interest that at 28 days postburned, esophagus sections from 3-AB–treated rats appear more normal than those of untreated rats and also confirmed this protective effect of 3-AB. Esophageal strictures caused by ingestion of corrosive materials are a major clinical problem with a high degree of morbidity. Most clinicians still use the tedious practice of recurrent esophageal dilatations, facing the problem of recurrent fibrosis and stricture after the dilatation. Several lines of evidence indicate that ROS are generated at the burn site immediately after injury [4-6,22]. Gunel et al
Table 3
[4,5] have shown that tissue oxygen radical levels are significantly increased and antioxidant capacity is decreased 72 hours after caustic injury. And it is well established that cellular DNA damage results from the generation of reactive radicals. The regenerative capacity of tissue immediately after injury is dependent on the number of noninjured or sublethally injured cells that survive and can initiate the reparative mechanisms that restore the structure of the tissue [23]. One of the major requirements for initiation of the cellular repair process is the repletion of intracellular ATP [6,24,25]. On the other hand, extensive DNA damage after injury because of generated oxygen radicals may lead to excessive PARP activation that consumes large quantities of cellular nicotinamide adenine dinucleotide resulting in ATP depletion and dysfunction or necrotic cell death [24]. Although we did not determine whether PARP inhibition increased levels of tissue ATP postexposure to NaOH, our data showed that PARP inhibition restores cell injuries. This is consistent with decreased level of MDA, PCC (indices of tissue damage), HP, and ameliorated histopathologic findings in our work. We found that tissue SOD and GPx enzyme activity were decreased in untreated rats, whereas those were increased in rats that received 3-AB. Moreover, tissue MDA and PCC levels were found significantly increased in the untreated
Comparison of biochemical evaluations
Sham-operated group (n = 15) Untreated group (n = 9) Treatment group (n = 13)
HP (μg/mg tissue)
SOD (U/mg protein)
GPx (U/mg protein)
MDA (μmol/g protein)
PCC (μmol/g protein)
1.4 ± 0.2
21.3 ± 1.2
33.2 ± 1.8
0.30 ± 0.02
0.39 ± 0.01
3.1 ± 0.6 ⁎ 1.7 ± 0.3
10.4 ± 1.0 ⁎ 20.1 ± 1.7
22.1 ± 1.8 ⁎ 31.5 ± 2.8
0.55 ± 0.03 ⁎ 0.35 ± 0.03
0.59 ± 0.02 ⁎ 0.45 ± 0.02 †
⁎ Statistically significant from sham-operated and treatment group. † Statistically significant from sham-operated group.
1478 group and decreased in the treated group. The antioxidant systems protect the cell against lipid peroxidation and protein oxidation. These results consistent with reactive oxygen radicals play an important role in caustic esophagus. Gunel et al [4] reported that MDA level was increased, and glutation level (antioxidant substance that augments glutathione GPx activity) was decreased at 24 hours and remained 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, and catalase were decreased at 28 days in corrosive esophagitis in rats. We can conclude that caustic esophagitis is associated with increased reactive oxygen radicals and tissue damage. In addition to this, 3-AB may be a possible beneficial agent to reduce tissue damage by enhanced antioxidant enzyme activity. One could argue that the beneficial effects of 3-AB are because of its ability to scavenge free radicals. There is evidence that the PARP inhibitors scavenge reactive oxygen radicals. This notion is supported by our finding that PARP inhibitor was able to ameliorate the tissue SOD and GPx enzyme activity determined to evaluate antioxidant capacity of the tissue. To date, there has been no research into the role of PARP within the esophagus under normal and pathophysiologic conditions. However, several experimental studies showed that the inhibition of PARP-activation pathway ameliorates the tissue injury because of free radicals in various forms of shock, inflammation, trauma, and reperfusion injury [23,24,27,28]. From this study, it can be concluded that 3-AB have antioxidant effect in caustic esophageal burn in rats. In the present study, administration of 3-AB during the first 5 days after caustic injury significantly decreased the HP level, SI, and histopathologic injury score compared with the untreated rat. Collagen deposition in the submucosa and damage to the muscularis mucosa and tunica muscularis in treated group were less than that in untreated group. These histopathologic findings showed correlation with biochemical results. It is well known that alkaline caustic agents are destructive to the esophagus by a process of liquefaction necrosis [3,9]. Therefore, preventing stricture formation should be supporting normal wound healing in esophageal wall at early phase. It was seen that PARP-inhibitor treatment significantly reduced neuronal [29], retinal [30], and heart or skeletal muscle [28]. Furthermore, 3-AB decreased progression of postinfarction heart interstitial fibrosis [28,31]. Taken together, it might be possible to prevent smooth muscle death by prevention of ATP depletion in cells, which decreased the fibroplasia during healing of corrosive esophageal burns by using 3-AB. The starting time of medication in the studies related to PARP inhibitors is generally before the injury [24,27,30]. In contrast to these studies, we started with treatment with 3-AB immediately after the burn was created. After exposure, victims, especially children, can arrive to the emergency department in a few hours. Because metabolic rate of
A. Guven et al. rodents, especially rats, is almost 10-fold faster than that of humans, to start medication in a few minutes after injury seems to be realistic. Nevertheless, time-course studies are necessary to determine the starting time after the injury. It is also necessary to design further studies to clarify pathophysiologic mechanism of caustic burn and to determine whether the beneficial effect of this agent is irreversible. Inhibition of PARP, especially chronic inhibition, may be harmful because this enzyme facilitates DNA repair that may be required in the recovery of esophagus in later phase. For this reason, determination of the proper timing for PARP inhibition postburned period may be a crucial factor to permit the use of PARP inhibitors in caustic esophageal burn. However, whatever the basis for beneficial action of 3-AB may be, our findings provide a rationale for using of inhibitors of PARP to ameliorate stricture formation because of caustic burn in humans. In conclusion, our results strongly showed that PARP plays an important role in the healing process in caustic esophagitis, and as a PARP inhibitor, 3-AB might be a potential agent to treat esophageal caustic burn by increasing antioxidant defense mechanism, decreasing tissue damage, and ameliorating histopathologic injuries. Further studies should be assessed to determine the side effect of this agent.
Acknowledgment We thank Dr Gokhan Gundogdu for the technical assistance during the experimental procedures.
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