Journal of Pediatric Surgery (2011) 46, 1893–1897
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The efficacy of single-dose 5-fluorouracil therapy in experimental caustic esophageal burn Levent Duman a,⁎, Behcet Ilker Büyükyavuz a , Irfan Altuntas b , Alpaslan Gökcimen c , Levent Ceyhan a , Hakan Darici c , Firdevs Aylak b , Onder Tomruk d a
Department of Pediatric Surgery, Süleyman Demirel University Medical School, 32260, Isparta, Turkey Department of Biochemistry, Süleyman Demirel University Medical School, 32260, Isparta, Turkey c Department of Histology and Embryology, Süleyman Demirel University Medical School, 32260, Isparta, Turkey d Department of Emergency, Süleyman Demirel University Medical School, 32260, Isparta, Turkey b
Received 27 January 2011; revised 21 February 2011; accepted 2 March 2011
Key words: Caustic esophageal burn; Esophageal stricture; 5-fluorouracil
Abstract Introduction: Accidental ingestion of caustic substances may cause serious problems in children. Approximately 20% of caustic ingestions result in esophageal stricture formation, resulting from excessive collagen synthesis to the extracellular matrix by fibroblasts. Recent studies showed that a single application of 5-fluorouracil (5-FU) is a very effective inhibitor of fibroblast proliferation and differentiation for prolonged periods. Using an experimental model, we investigated the efficacy of single-dose 5-FU on stricture formation after caustic esophageal burn. Materials and methods: Forty Wistar-Albino rats were divided randomly into 4 equal groups: group 1 (sham-operated group), the esophagus was uninjured and untreated; group 2 (control group), the esophagus was injured and left untreated; group 3 (intraperitoneal treatment group), the esophagus was injured and treated immediately after the burn injury with a single intraperitoneal dose (20 mg/kg) of 5-FU; group 4 (local treatment group), the esophagus was injured and treated immediately after the burn injury with a single intraesophageal application of 5-FU at a concentration of 25 mg/mL. Caustic esophageal burn was produced by instilling 10% NaOH in the distal esophagus. The distal esophagi were harvested at 28 days postoperatively. Histologic sections were assessed by measuring the stenosis index (SI) and histopathologic damage score. Hydroxyproline (HP) levels in the tissues were determined biochemically. Results: There were significant reductions in the SI (P b .05), histopathologic damage score (P b .05), and HP level (P b .05) in the intraperitoneal treatment group when compared with the control group. No significant differences in the SI and histopathologic damage score were detected between the control and local treatment groups (P N .05), whereas significant reduction in the HP level was determined between these groups (P b .05). Conclusion: A single intraperitoneal dose of 5-FU had a preventive effect on stricture formation after caustic esophageal burn. This observation suggests that 5-FU may prevent this undesirable complication in the clinical setting. Clinical studies are now required to verify this form of treatment. Local intraesophageal application of 5-FU immediately after the burn injury was not effective. Further investigations are required to determine the appropriate timing of application of 5-FU at the local site of injury. © 2011 Elsevier Inc. All rights reserved.
⁎ Corresponding author. Tel.: +90 2462119249; fax: +90 2462371758. E-mail address:
[email protected] (L. Duman). 0022-3468/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2011.03.001
1894 Accidental ingestion of caustic substances represents a medical challenge in childhood. Approximately 20% of caustic ingestions result in esophageal stricture formation [1]. Several types of nonsurgical management such as steroids, antibiotics, esophageal dilatation, and esophageal stents have been proposed for this complication [2,3]; but current forms of treatment are relatively ineffective for severe corrosive burns. Although caustic esophageal burn is one of the most studied experimental models, most of the suggested drugs to prevent complications are less than ideal. A majority of the proposed agents require multiple doses, meaning longer hospitalization and some potential undesirable side effects in clinical practice. Esophageal stricture formation is the result of excessive collagen synthesis to the extracellular matrix by fibroblasts. In theory, any intervention that leads to cessation of collagen synthesis could be used to prevent stricture formation. A single application of 5-fluorouracil (5-FU) is a very effective inhibitor of fibroblast proliferation and differentiation for prolonged periods [4]. We theorized that 5-FU might prevent stricture formation after caustic ingestions. In this experimental study, we investigated the efficacy of single-dose 5-FU therapy on stricture formation after a caustic esophageal burn.
1. Materials and methods This study was approved by the Experimental Ethics Committee of Süleyman Demirel University Medical School, Isparta, Turkey.
1.1. Study groups The study was performed using 40 Wistar-Albino rats weighing between 180 and 240 g. They were randomly and equally divided into 4 groups: group 1 (sham-operated group), the esophagus was uninjured and untreated; group 2 (control group), the esophagus was injured and left untreated; group 3 (intraperitoneal treatment group), the esophagus was injured and treated with a single intraperitoneal dose (20 mg/kg) of 5-FU immediately after the burn injury; group 4 (local treatment group), the esophagus was injured and treated with a single intraesophageal application of 5-FU at a concentration of 25 mg/mL immediately after the burn injury.
1.2. Surgical procedure and sample collection After 12 hours of fasting, rats were anesthetized intraperitoneally with xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (50 mg/kg). The method described by Gehanno and Guedon [5] was used to create caustic esophageal burns. Using sterile surgical techniques, a
L. Duman et al. midline laparotomy was performed; and 1.5-cm segment of the abdominal esophagus was isolated. A 24-G cannula was placed into the isolated segment through a gastric puncture, and esophageal segment was tied with 2-0 silk sutures distally and proximally to prevent leakage of the caustic agent. Ten percent NaOH solution was instilled through the cannula for 3 minutes, then the solution was aspirated. Subsequently, distilled water was used to irrigate the burned area segment for a 60-second period. In the sham-operated group, the distal esophagus segments were irrigated with 0.09% NaCl solution only, whereas 20 mg/kg 5-FU was given intraperitoneally after this process in the intraperitoneal treatment group. In the local treatment group, 5-FU solution at a concentration of 25 mg/mL was instilled into the isolated esophageal segment for 5 minutes; and it was rinsed with distilled water. The proximal and distal sutures were cut, and the cannula was pulled back after these instillations. The abdominal incision was closed, and 10-mL 0.09% NaCl solution was administered subcutaneously to all animals. The rats were not allowed to feed for the next 24 hours, then they were fed with standard rat chow and water ad libitum during the experimental period. On the 28th postoperative day, the rats were killed; and distal 1.5-cm esophageal segments of each animal were harvested and transversally sectioned into 2 equal parts. Proximal portions were placed into 10% neutral formaldehyde solution for histologic evaluation. The distal portions were stored at −80°C until assayed for biochemical evaluation.
1.3. Histopathologic evaluation Histopathologic analysis was performed by the same observer in a blind manner. Tissue sections from routinely embedded paraffin blocks were stained with hematoxylin and eosin and Masson trichrome and evaluated by light microscopy using the stenosis index (SI) and histopathologic damage score. The thickness of the esophageal wall and the lumen diameter were measured to calculate the SI (SI = wall thickness/lumen diameter). In addition, histopathologic damage was scored according to a scale in 3 categories, collagen deposition in the submucosa, damage to muscularis mucosa, and damage and collagen deposition in the tunica muscularis, for a total score of 0 to 5 (Table 1).
1.4. Tissue hydroxyproline assay This biochemical assay was performed by the same observer in a blind manner. Hydroxyproline (HP) level was calculated in dry tissue samples using the partially modified method of Woessner [6].
1.5. Statistical analysis All statistical analyses were performed using SPSS software for Windows (release 15.0; SPSS, Inc, Chicago,
Efficacy of single-dose 5-FU caustic esophageal burn Table 1
Histopathologic evaluation criteria
Criteria
Score
Collagen increase in submucosa None Mild Significant 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 fibers)
0 1 2 0 1 0 1 2
IL). All data were presented as mean ± SD of the mean. All parameters were analyzed by 1-way analysis of variance with the Tukey multiple comparison test. P b .05 was considered significant.
2. Results All animals in the sham-operated group survived during the study. Three control and 2 local treatment group rats died because of esophageal perforation. Two rats died because of evisceration in the intraperitoneal treatment group. Table 2 shows the SIs, HP levels, and histopathologic scores (mean ± SD) of the groups.
Fig. 1 Esophageal section of the control group shows marked increase in submucosal and tunica muscularis collagen content and damage to muscularis mucosa (hematoxylin and eosin stain, original magnification ×4 and ×10).
2.2. Histopathologic damage score The histopathologic damage score was significantly higher in the control group (Fig. 1) compared with the sham-operated (Fig. 2) and intraperitoneal treatment groups (P b .05), whereas no significant difference was found between the local treatment and control groups (P N .05). In the intraperitoneal treatment group, there was a mild increase in the submucosal and tunica muscularis collagen accumulation (Fig. 3), whereas it was more pronounced in the local treatment group (Fig. 4).
2.3. Tissue HP level
2.1. Stenosis index The SI in the control group was significantly higher compared with the sham-operated and intraperitoneal treatment groups (P b .05). Although the SI in the local treatment group decreased compared with the control group, this was not statistically significant (P N .05). Table 2
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Hydroxyproline level in the control group was significantly higher when compared with the other 3 groups (P b .05).
Results and comparison of the study groups
Sham-operated group (n = 10) Control group (n = 7) IP treatment group (n = 8) Local treatment group (n = 8)
SI
Histopathologic HP (μg/mg damage score tissue)
0.59 ± 0.24
0
43.04 ± 9.30
1.07 ± 0.37 a 3.14 ± 0.89 a
80.83 ± 26.30 b
0.64 ± 0.29
1.37 ± 0.74
45.71 ± 12.53
0.70 ± 0.30
2.12 ± 0.99
55.09 ± 15.79
IP indicates intraperitoneal. a Statistically significant from the sham-operated and intraperitoneal treatment group. b Statistically significant from the other groups.
Fig. 2 Normal esophageal appearance in the sham-operated group (hematoxylin and eosin stain, original magnification ×4).
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Fig. 3 Esophageal section of the intraperitoneal treatment group shows mild increase in submucosal and tunica muscularis collagen content (hematoxylin and eosin stain, original magnification ×10).
3. Discussion Accidental ingestion of caustic substances is an important childhood health problem with a high degree of morbidity. Extensive damage to the esophagus can cause serious problems such a stricture formation. The treatment of this long-term complication continues to be a major concern for pediatric surgeons. Fibroblasts play a chief role in the healing process. The role of fibroblasts involves the synthesis of proteoglycan and collagen fibers [7]. Although the synthesis of collagen is important for reestablishment of structural integrity, the same mechanisms participate in the development of stricture formation after caustic injury. It seems logical that medications targeting cessation of collagen synthesis by fibroblasts may be useful in preventing this fibrosisrelated complication. 5-FU is one of the most commonly used antineoplastic drugs. The principal therapeutic use of this drug is as a single agent or in combination chemotherapy for the treatment of breast, gastrointestinal, and ovarian cancer [8]. 5-FU is an antimetabolite that specifically mediates its antiproliferative effect by interfering with pyrimidine metabolism, which is a nucleotide required for DNA replication [9]. Recent studies showed that a single dose of 5-FU is a very effective inhibitor of fibroblast proliferation and differentiation for prolonged periods [10,11]. The putative mechanism of 5-FU action, inhibition of thymidylate synthetase, and impairing function of nucleic acids would lead to a global inhibition of fibroblast function [12]. These observations have stimulated great interest in the potential benefits of this antimetabolite in fibrosis-related disorders. For example, it has been successfully used clinically in the prevention of scar tissue formation in the eye after filtration surgery for glaucoma.[13,14]. Furthermore, a variety of experimental studies have been conducted to evaluate the effectiveness of 5-FU on distinct
L. Duman et al. pathologies, such as tendon adhesions after tendon repairs, scar development after skin incisions, and capsule formation around breast implants [15-17]. To the best of our knowledge, there have been no studies examining the efficacy of 5-FU on stricture formation after caustic esophageal injury. Esophageal stricture formation after caustic injury occurs at approximately 21 days and is completed from 28 to 42 days [18]. Thus, preventive treatment should be effective during this period. Occleston et al [4] investigated the long-term effect of 5-FU on collagen type I and fibronectin synthesis by ocular fibroblasts and found that exposure to 5-FU (0.25-25 mg/mL) for 5 minutes led to decrease in the extracellular matrix throughout the experimental period of 48 days. Another study demonstrated that extremely short exposures of ocular fibroblasts to antiproliferative agents resulted in growth arrest for prolonged periods of at least 36 days in tissue culture [10]. A single-dose of 5-FU was used in this study. In the present experiment, we used intraperitoneal 5-FU at a dose of 20 mg/kg, which is the highest nonlethal dose of this antimetabolite for rats [19]. Bulstrode et al [12] evaluated the effects of a single 5-minute exposure of different 5-FU concentrations on tendon fibroblasts and found that 5-FU caused a dose-dependent decrease in collagen synthesis, and this effect was maximal at a concentration of 25 mg/mL. Therefore, we used this concentration in the local treatment group. More recently, 5-FU has been shown to selectively reduce collagen type I synthesis [12]. Fibrosis mainly comprises collagen type I; therefore, selective inhibition of this type collagen synthesis would reduce stricture formation without affecting tissue structure. In our study, improvement of the esophagus without significant stenosis in the intraperitoneal treatment group may be associated with this selective collagen inhibition. Toxic side effects are the major dose-limiting factors of chemotherapeutic agents. Any therapy attempting to prevent stricture formation should not cause significant complications.
Fig. 4 Esophageal section of the local treatment group shows moderate increase in submucosal and tunica muscularis collagen content (hematoxylin and eosin stain, original magnification ×10).
Efficacy of single-dose 5-FU caustic esophageal burn 5-FU is a cytotoxic agent that has some significant side effects in clinical practice. This antimetabolite leads to DNA damage and cell death only in high doses [10]. At much lower doses, 5-FU has no cytotoxic effects but only decreases cellular metabolism and acts as a negative regulator of collagen synthesis, causing a significant reduction in collagen protein synthesis by fibroblasts [12]. It has also been shown that cells that are growth arrested by 5-FU at much lower doses are still capable of certain functions, such as migration and secretion, which give them the capacity to be involved in healing [4,11]. Some previous studies showed that colonic anastomotic healing was impaired by 5-FU [20,21], suggesting that the agent may have a negative influence on wound healing. In our study, 2 rats in the intraperitoneal treatment group had evisceration, which may be the result of reduced collagen synthesis in the wound or more likely a technical problem with wound closure. We believe that these instances of evisceration were a result of a technical problem because eviscerations occurred within the first few days after the surgical procedure. Based on these observations, we propose that 5-FU can be used in clinical practice to prevent stricture formation after caustic esophageal burns without causing any significant side effects. Collagen is the main product of fibroblasts. Tissue HP levels are the best indicator of collagen synthesis. Hydroxyproline is an amino acid and a subproduct of collagen synthesis, which shows a parallel increase with collagen synthesis [22]. In our study, the tissue HP level of the intraperitoneal treatment group was significantly decreased compared with the control group. This biochemical finding also correlated with the SI and histopathologic damage score showing collagen deposition in the submucosa and tunica muscularis. These results suggest that a single intraperitoneal dose of 5-FU has a preventive effect on stricture formation after caustic esophageal burn. The HP level of the local treatment group was significantly decreased, but this finding showed no correlation with the histologic parameters. In our study, 5-FU was applied immediately after the burn injury in the local treatment group. As a result, we think that direct application on the damaged esophagus during the acute necrotizing phase decreases the effectiveness of 5-FU and, perhaps, more successful results may be obtained by later applications. In this regard, further investigations are needed to determine the most effective application time of 5-FU at the local injury site. A single–intraperitoneal dose 5-FU has a preventive effect on stricture formation after an experimental caustic esophageal burn. This observation suggests that it may be useful in preventing this undesirable complication in clinical practice. Clinical studies are now required to verify this form of treatment. Local application of 5-FU immediately after the burn injury was ineffective. Further investigations are needed to determine the appropriate timing for intraesophageal application of 5-FU at the injured site.
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