Pathophysiology 13 (2006) 23–27
Antireflux surgery and esophageal mucosal DNA damage Niku K.J. Oksala a,b,c,∗ , Mustafa Atalay c , Tuomo K. Rantanen d a
Department of Surgery, Tampere University Hospital, Tampere, Finland Department of Surgery, Kuopio University Hospital, Kuopio, Finland c Department of Physiology, University of Kuopio, Finland Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland b
d
Received 1 October 2005; received in revised form 6 October 2005; accepted 6 October 2005
Abstract Gastroesophageal reflux disease (GERD) is characterized by reflux of gastroduodenal contents, esophagitis and oxidative tissue damage in the distal esophagus. It may ultimately lead to the development of a pre-malignant Barrett’s esophagus and subsequently to carcinoma. Antireflux surgery is an effective therapeutic tool to relieve GERD symptoms and to normalize the reflux to the distal esophagus. However, antireflux surgery may be insufficient to restore oxidative insult, which can promote DNA adduct formation and subsequent initiation of carcinogenesis. Controversy exists whether antireflux surgery can reverse the development of carcinoma in the mucosa. We aimed to test the effect of antireflux surgery on DNA adduct formation in the esophagus. Patients (n = 19) with objectively confirmed GERD underwent antireflux surgery and were followed up for 6 months after which a symptom evaluation, control endoscopy, biopsy and pH-measurements were performed. The amounts of DNA adducts in the proximal and distal mucosa of the esophagus were measured using the 32-P-postlabelling method. After the surgery, esophageal acid exposure was normalized in all the patients and symptoms were relieved in all but one patient. Endoscopic examinations showed that erosive esophagitis had healed in all the cases 6 months after the surgery. Barrett’s esophagus was found in six cases in preoperative biopsies. The amount of DNA adducts in the distal esophagus was higher than in the proximal esophagus both pre- and postoperatively. Antireflux surgery did not change this pattern and was thus not capable of reducing DNA adduct formation. The level of DNA damage was similar in the patients having Barrett’s esophagus compared to the rest of the patients. Antireflux surgery is insufficient to normalize DNA damage due to GERD. Our observations suggest that antireflux surgery is perhaps not effective in the prevention of carcinogenesis because of the persisting DNA damage. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Gastroesophageal reflux; Barrett’s esophagus; DNA adducts; Antireflux surgery
1. Introduction The incidence of esophageal adenocarcinoma has risen dramatically in western countries in recent decades [1,2]. The causes of adenocarcinoma of the esophagus are poorly understood. The symptomatic gastroesophageal reflux disease (GERD) resulting in pathological reflux of gastroduodenal contents to distal esophagus has, however, been found to be one of the reasons for the esophageal adenocarcinoma [3]. Barrett’s esophagus is a pre-malignant condition of the distal esophagus characterized by stepwise progression from metaplasia and dysplasia to cancer as a consequence of GERD ∗
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[4]. Fundoplication is a surgical procedure, which eliminates reflux and is indicated in patients with GERD unresponsive to medical treatment. Despite fundoplication is effective in minimizing fluxes of gastric juice and duodenal contents to the distal esophagus, there is increasing concern whether this operation is truly effective as an antineoplastic procedure to prevent the transformation of normal mucosa to a dysplastic one and of Barrett’s esophagus to a cancer [5–10]. Oxidative stress has been defined as an imbalance of the pro-oxidant/antioxidant equilibrium in favor of the prooxidants. Oxidative stress plays a role in esophageal cancer [11]. High concentrations of reactive oxygen species (ROS) are hazardous for organisms and they damage all major cellular constituents. In biological systems, cells respond to mild oxidative stress by inducing their antioxidant defenses
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and other protective systems, but severe oxidative stress can cause permanent damage to DNA, proteins and lipids. Oxidative stress may also cause irreversible modifications of DNA. DNA adduct formation initiates carcinogenic processes and detection of DNA adducts may be used to assess the risk level of carcinogenesis [12]. GERD, Barrett’s esophagus and adenocarcinoma are characterized by increased amount of DNA adducts in the distal esophagus, and it is paralleled by increased oxidative stress [13–15]. Oxygen free radicals are highly reactive and capable of inducing formation of DNA adducts [16]. We have previously observed that fundoplication is not capable of normalizing oxidative stress in the distal esophagus [17]. This raises serious doubts whether antireflux surgery has any effect on DNA adducts, the ultimate manifestation of oxidative stress and initiation of carcinogenesis [12,16]. Since a substantial amount of evidence indicates that fundoplication is not sufficient to prevent carcinogenesis in the distal esophagus [5–10], we hypothesized that it does not reduce DNA adduct formation. To test this hypothesis, DNA adduct formation was measured in patients with GERD who underwent fundoplication, both pre- and postoperatively in the proximal and distal esophagus. 2. Materials and methods 2.1. Patients Initially, 20 consecutive patients with GERD who underwent Nissen–Rossetti fundoplication in Kanta–H¨ame Central Hospital during year 1998 were recruited in the study. One patient was dropped from the study because of insufficient biopsy material. Preoperatively, all patients had objectively verified GERD, including symptoms and abnormal esophageal 24-h pH-monitoring (median pH < 4 15.7%; range 5.2–55.4%) [18]. Upper gastrointestinal endoscopy and esophageal manometry were preoperatively performed to every patient. Using Savary–Miller’s grading system [19] endoscopy had revealed no macroscopic esophagitis (grade 0) in eight patients (40%), grade I esophagitis in one patient (5%), grade II in four patients (20%) and grade IV in seven patients (35%; Barrett’s esophagus in six and esophageal ulcer in one). In all cases, medical treatment with proton pump inhibitors (PPIs; 18 patients) or H2-blockers (two patients) had failed or patients were dependent on these medicines and requested surgical treatment. All medicines used for GERD were withdrawn 2 weeks before the operation. All patients were interviewed using DeMeester–Johnson reflux scale [18]. 2.2. Surgical procedures Nissen–Rosetti fundoplication was performed by fashioning a floppy 360◦ posterior 2-cm long fundic wrap over a 48-Fr esophageal bougie. The technique included posterior crural repair, and the wrap was anchored to the cardia with
a non-absorbable braided polyester suture [20]. No postoperative mortality occurred. At the beginning of the operation upper gastrointestinal endoscopy was performed and six biopsies from the distal esophagus (four for the determination of DNA adducts and two for histological examination) and 6 biopsies from the proximal esophagus 20 cm from the incisor level were taken using Olympus FB-53U-1 biopsy forceps. Biopsies were taken from macroscopic lesions and if there was no lesion, 0–5 cm above the esophagogastric junction. In patients with Barrett’s esophagus, biopsies were taken from the area of Barrett’s metaplasia. Biopsies for determination of DNA adducts were immediately frozen and stored at −70 ◦ C. Biopsies for the histological examination were fixed in 10% buffered formalin solution. Six months after the operation endoscopy, pH-measurements and biopsies were taken from the proximal and distal esophagus using the same criteria as preoperatively. The Ethics Committee of Kanta–H¨ame Central Hospital approved this protocol. All the patients involved gave their informed consent. 2.3. DNA adduct analysis Tissue samples were homogenized under liquid nitrogen using a Mikro-Dismembrator (Braun, Melsungen, Germany). DNA isolation was performed as earlier described [21]. DNA was digested enzymatically to 3 -mononucleotides [22]. DNA was incubated for 2 h at 37 ◦ C with micrococcal nuclease (80 mU/g DNA in 3 mM bicine pH 9.0, 0.5 mN CaCl2 , then for 2 h at 37 ◦ C with spleen phosphodiesterase (1.6 mU/g DNA) in added 20 mM ammonium acetate, pH 5.0. The modified nucleotides were converted to 32-P-postlabelled diphosphates in a mixture (2 l) containing 2.4 U T4 polynucleotide kinase and ATP (7 Ci, 3000 Ci/mmol). Labelling was carried out at pH 9.6. TLC analyses were carried out on 10 × 15 cm PEI-cellulose TLC plates (Macherey-Nagel, Duren, Germany) [22]. The plates were first developed (D1) with 1.7 M NaH2 PO4 (pH 6.0) using a paper wick on top. The upper part of plates with the wick was cut off and discarded. The plates were then developed in the opposite direction (D3) with 2.7 M lithium formate and 6.4 M urea (pH 3.5). The next direction (D4) was developed at 90◦ from D3 in 0.4 M lithium chloride, 0.25 M Tris–HCl, 4.25 M urea (pH 8.0) into a paper wick, which was then cut off. D5 was developed into the same direction as D4 in 1.7 M NaH2 PO4 (pH 6.0) with a paper wick. After each direction was completed, the plates were washed by flotation in water and dried. The adducts were detected and quantified using a Bio-Rad Image Analysis System (Bio-Rad Laboratories, Hercules, CA, USA). The average levels of DNA adducts are expressed as adducts/109 nucleotides. 2.4. Statistical methods Median and range were provided as descriptive statistics of the data. Wilcoxon signed ranks test for paired
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and Mann–Whitney U-test for unpaired samples was used to analyze the differences in DNA adduct levels and pH < 4 values between preoperative and postoperative biopsy specimen. A probability of 0.05 was accepted as significant.
3. Results 3.1. Effect of fundoplication on GERD
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Table 2 The effect of fundoplication in non-Barrett and Barrett’s esophagus: DNAadducts in the esophageal mucosa before and 6 months after the fundoplication in gastroesophageal reflux patients Non-Barrett (N = 13) Proximal Preoperative Postoperative
6.9 (2.2–12.3) 7.8 (0.9–25.2)
Distal Preoperative Postoperative
10.8 (4.6–22.5)* 11.1 (2.2–30.6)*
Barrett (N = 6) 8.2 (5.3–11.6) 13.1 (6.2–21.6) 12.1 (10.0–15.8)* 20.7 (4.3–35.3)*
*
After 6 months, one patient had upper abdominal pain symptoms, but had a normal fundic wrap at endoscopy and normal acid exposure according to postoperative 24-h pH measurement. The percentage proportion of pH below 4 was significantly lower postoperatively compared to preoperative values (p = 0.001). In the whole study group, esophageal acid exposure was normalized in all patients 6 months after the fundoplication. Endoscopy showed that erosive esophagitis had healed in all cases. Barrett’s esophagus was found in six cases as was also the case in preoperative biopsies. 3.2. Effect of fundoplication on DNA adducts There was no difference in the amount of DNA adducts between preoperative and postoperative biopsies either in the distal (p = 0.334) or in the proximal esophagus (p = 0.171) (Table 1). The amount of DNA adducts was higher in the distal than in the proximal esophagus both preoperatively (p = 0.001) and postoperatively (p = 0.016) (Table 1). 3.3. Effect of Barrett’s esophagus on DNA adducts In subgroup analysis comparing subjects with Barrett’s esophagus to subjects without it, in proximal esophagus there was no difference either pre- (p = 0.188) or postoperatively (p = 0.096) (Table 2). Similarly, in distal esophagus there was no difference either pre- (p = 0.292) or postoperatively (p = 0.096) (Table 2). The amount of DNA adducts was higher in the distal than in the proximal esophagus both preoperatively (p = 0.01) and postoperatively (p = 0.01) (Table 2). Table 1 The effect of fundoplication: DNA-adducts in the esophageal mucosa before and 6 months after the fundoplication in gastroesophageal reflux patients
Proximal Distal
Preoperative (N = 19)
Postoperative (N = 19)
7.4 (2.2–12.3) 11.0 (4.6–22.5)***
7.8 (0–25.2) 12.4 (2.2–35.3)*
* p < .05 between proximal and distal. Median (minimum–maximum) adducts/109 . *** p < .001 between proximal and distal. Median (minimum–maximum) adducts/109 .
p < .05 between proximal and distal. Median (minimum-maximum) adducts/109 nucleotides.
4. Discussion In the present study, we showed that fundoplication, which was effective in normalizing the acid reflux, symptoms and erosive esophagitis in GERD-patients, did not have any effect on the espohageal mucosal DNA-damage as indicated by unchanged DNA adducts after a follow-up for 6 months. We showed that in all patients fulfilling the criteria for GERD, there was an increased amount of DNA adducts in the distal esophagus compared to the proximal esophagus before as well as 6 months after the surgery. The amount of DNA adducts was similar in the patients with Barrett’s esophagus as well in the rest of the patients who did not have Barrett’s dysplasia. Our results indicate for the first time that fundoplication is insufficient to restore the DNA damage in the distal esophagus. The diagnosis was confirmed with biopsy, manometry and pH-measurements. Our method of fundoplication was considered to be effective, because the pH-measurements and endoscopic erosive esophagitis were normalized by the operation, which also provided relief from GERD symptoms in all except one patient. The presence of Barrett’s transformation and mild dysplasia remained unchanged after surgery. Our results are in accordance with those observed earlier [18,23]. We found a constant pattern of increased DNA adducts in the distal esophagus compared to proximal esophagus in all the patients pre- and postoperatively, which was unaffected by the fundoplication. These findings suggest that although the acid reflux and erosive esophagitis were normalized, the surgical process we used had no curative effect against DNA damage in the distal esophagus. This observation has not been reported before and no similar data exists in the literature available to be compared with our results. In subgroup analysis comparing the subjects having Barrett’s esophagus with the subjects having no Barret’s esophagus, there was no difference in DNA adducts in the proximal or distal esophagus either pre- or postoperatively. The degree of dysplasia had no effect on the amount of DNA adducts. Previously, in GERD patients with disease ranging from esophagitis and Barrett’s esophagus to adenocarcinoma there
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was a constant pattern of increased DNA adducts in the distal esophagus with highest levels in Barrett’s esophagus and significantly lower levels in adenocarcinoma [13–15]. In the present study, because of the limited amount of patients, it was not possible to analyze the effect of different dysplasia gradus on DNA adducts. The reason for DNA adduct formation in the distal esophagus is not clear. Increased oxidative stress and oxidative tissue injury have been demonstrated in patients with GERD ranging from esophagitis to Barrett’s esophagus and adenocarcinoma [24,14,15]. In GERD patients, increased levels of myeloperoxidase and DNA adducts and decreased levels of potent antioxidant, total glutathione was observed [14]. This finding is of paramount significance, since oxygen free radicals are capable of inducing formation of DNA adducts [16]. It is probable that in GERD there is a subpopulation of transformed cells with defective defense mechanisms against DNA-damage, which could be responsible for constant presence of DNA adducts despite the surgery. Our results are supported by the finding that long-lasting acid suppression therapy does not alter malignant transformation of Barret’s esophagus, which seems to be attributed to defects in DNA repair and cell cycle control [25]. However, because of the small number of patients and relatively short duration of follow-up, we still need to be cautious to interpret the outcome of the present study.
5. Conclusion Our results of persisting high DNA adduct levels may indicate that fundoplication is not capable of normalizing the DNA-damage in the distal esophagus. More prospective data are needed to clarify whether this is a reason why fundoplication seems to be ineffective preventing the development of esophageal carcinoma.
Acknowledgements This work has been supported by the grants from the Finnish Ministry of Education, Mary and Georg C. Ehrnrooth Foundation, Yrj¨o Jahnsson Foundation, Helsinki, Finland, and Fund for Advanced Technology of Eastern Finland.
References [1] M. Pera, A.J. Cameron, V.F. Trastek, H.A. Carpenter, A.R. Zinsmeister, Increasing incidence of adenocarcinoma of the esophagus and esophagogastric junction, Gastroenterology 104 (1993) 510– 513. [2] E.I. Sihvo, J.T. Salminen, O.J. Ramo, J.A. Salo, The epidemiology of oesophageal adenocarcinoma: has the cancer of gastric cardia an influence on the rising incidence of oesophageal adenocarcinoma? Scand. J. Gastroenterol. 35 (2000) 1082–1086.
[3] J. Lagergren, R. Bergstrom, A. Lindgren, O. Nyren, Symptomatic gastroesophageal reflux as a risk factor for esophageal adenocarcinoma, N. Engl. J. Med. 340 (1999) 825–831. [4] D. Levine, Management of dysplasia in the columnar-lined esophagus, Gastroenterol. Clin. North Am. 26 (1997) 613–634. [5] K.E. Corey, S.M. Schmitz, N.J. Shaheen, Does a surgical antireflux procedure decrease the incidence of esophageal adenocarcinoma in Barrett’s esophagus? A meta-analysis, Am. J. Gastroenterol. 98 (2003) 2390–2394. [6] P. Parrilla, L.F. Martinez de Haro, A. Ortiz, V. Munitiz, J. Molina, J. Bermejo, M. Canteras, Long-term results of a randomized prospective study comparing medical and surgical treatment of Barrett’s esophagus, Ann. Surg. 237 (2003) 291–298. [7] A.E. Abbas, C. Deschamps, S.D. Cassivi, M.S. Allen, F.C. Nichols III, D.L. Miller, P.C. Pairolero, Barrett’s esophagus: the role of laparoscopic fundoplication, Ann. Thorac. Surg. 77 (2004) 393– 396. [8] A. Csendes, Surgical treatment of Barrett’s esophagus: 1980–2003, World J. Surg. 28 (2004) 225–231. [9] P. Sharma, Barrett esophagus: will effective treatment prevent the risk of progression to esophageal adenocarcinoma? Am. J. Med. 117 (Suppl. 5A) (2004) 79S–85S. [10] N.J. Shaheen, Does fundoplication change the risk of esophageal cancer in the setting of GERD? Am. J. Gastroenterol. 100 (2005) 1009–1011. [11] X. Chen, C.S. Yang, Esophageal adenocarcinoma: a review and perspectives on the mechanism of carcinogenesis and chemoprevention, Carcinogenesis 22 (2001) 1119–1129. [12] E.R. Nestmann, D.W. Bryant, C.J. Carr, Toxicological significance of DNA adducts: summary of discussions with an expert panel, Regul. Toxicol. Pharmacol. 24 (1996) 9–18. [13] J.T. Salminen, O.J. Ramo, M.O. Ahotupa, M.A. Farkkila, J.A. Salo, Increased DNA adducts in Barrett’s esophagus and refluxrelated esophageal malignancies, Ann. Med. 34 (2002) 565– 570. [14] E.I. Sihvo, J.T. Salminen, T.K. Rantanen, O.J. Ramo, M. Ahotupa, M. Farkkila, M.I. Auvinen, J.A. Salo, Oxidative stress has a role in malignant transformation in Barrett’s oesophagus, Int. J. Cancer 102 (2002) 551–555. [15] E.I. Sihvo, T. Ruohtula, M.I. Auvinen, A. Koivistoinen, A.L. Harjula, J.A. Salo, Simultaneous progression of oxidative stress and angiogenesis in malignant transformation of Barrett esophagus, J. Thorac. Cardiovasc. Surg. 126 (2003) 1952–1957. [16] S. De Flora, A. Izzotti, K. Randerath, E. Randerath, H. Bartsch, J. Nair, R. Balansky, F. van Schooten, P. Degan, G. Fronza, D. Walsh, J. Lewtas, DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine, Mutat. Res. 366 (1996) 197–238. [17] T. Rantanen, J. R¨as¨anen, E. Sihvo, M. Farkkila, J. Salo, The impact of antireflux surgery on oxidative stress of esophageal mucosa caused by gastroesophageal reflux disease: four-year follow-up study, Am. J. Gastroenterol., 2006, in press. [18] T.R. DeMeester, L. Bonavina, M. Albertucci, Nissen fundoplication for gastroesophageal reflux disease. Evaluation of primary repair in 100 consecutive patients, Ann. Surg. 204 (1986) 9–20. [19] M. Savary, G. Miller, The Esophagus, Handbook and Atlas of Endoscopy, Verlag Gassman AG, Solothurn, Switzerland, 1978, pp. 135–139. [20] T.K. Rantanen, J.A. Salo, J.T. Salminen, I.H. Kellokumpu, Functional outcome after laparoscopic or open Nissen fundoplication: a followup study, Arch. Surg. 134 (1999) 240–244. [21] R. Gupta, Non-random binding of the carcinogen N-hydroxy-2acetylaminofluorene to repetitive sequences of rat liver DNA in vivo, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 6943–6947. [22] K. Hemminki, P. Widlak, S.M. Hou, DNA adducts caused by tamoxifen and toremifene in human microsomal system and lymphocytes in vitro, Carcinogenesis 16 (1995) 1661–1664.
N.K.J. Oksala et al. / Pathophysiology 13 (2006) 23–27 [23] J.A. Salo, J.T. Salminen, I.H. Kellokumpu, Functional outcome after laparoscopic or open Nissen fundoplication, Arch. Surg. 134 (1999) 240–244. [24] G.J. Wetscher, R.A. Hinder, D. Bagchi, P.R. Hinder, M. Bagchi, G. Perdikis, T. McGinn, Reflux esophagitis in humans is mediated by oxygen-derived free radicals, Am. J. Surg. 170 (1995) 552–556, discussion 556–557.
27
[25] N. Carlson, J. Lechago, J. Richter, R.E. Sampliner, L. Peterson, R.M. Santella, J.R. Goldblum, G.W. Falk, A. Ertan, M. Younes, Acid suppression therapy may not alter malignant progression in Barrett’s metaplasia showing p53 protein accumulation, Am. J. Gastroenterol. 97 (2002) 1340–1345.
