Bile acids (taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid) develop esophageal cancer in a rat model of duodenoesophageal anastomosis after total gastrectomy

Introduction

Gastrectomy is the most common gastrointestinal surgery in Japan. Thoracic esophageal cancer in patients who underwent surgery has a history of gastrectomy in 3–10% of cases (1). Whether post-gastrectomy conditions lead to esophageal carcinogenesis is controversial. Distal gastrectomy is a good model for clinically examining esophageal reflux. It has been reported that symptomatic duodenal gastric reflux is the most frequent symptom of post-gastric surgery and is present in 35% (2).

Gastrectomy causes truncal vagotomy (TV), widening of the angle of His and small gastric remnant reduction. TV causes a decrease in gastric motility. The widening of angle of His causes deterioration of lower esophageal sphincter (LES). A small remnant stomach cannot accommodate a large amount of stomach content. These factors facilitate reflux of duodenal juice into the esophagus after gastrectomy.

We retrospectively evaluated 153 patients (3) undergoing subtotal esophagectomy for thoracic esophageal cancer. Divided into two groups, gastrectomy group, non-gastrectomy group. Group 1: 14 patients undergoing gastrectomy and Group 2: 139 patients not undergoing gastrectomy. The esophageal cancer occupies most of the lower esophagus in the post-gastric resection group, while the non-gastric resection group often has the middle esophagus. The histologic subtype was squamous cell carcinoma (SCC) in both gastric resection group and non-gastric resection group. Changes in the esophageal mucosa due to persistent reflux of stomach and duodenal contents (including bile) to the lower esophagus may occur more frequently in patients undergoing gastrectomy than in the intact stomach.

This study examined whether reflux of duodenal juice to the esophagus is involved in esophageal carcinogenesis. We present the following article in accordance with the ARRIVE reporting checklist (available at http://dx.doi.org/10.21037/aoe-20-47).

Methods

We used 8-week-old male Wistar rats (250–300 g). We are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study complies with the ethical regulation of the use of laboratory animals and all experiments follow the animal experiment guideline in the Kindai University. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Kindai University (KAME-22-02).

Surgical procedure

Esophagoduodenal anastomosis (EDA) (n=27) (Figure 1) (4)

Figure 1 Esophagoduodenal anastomosis with total gastrectomy model (EDA rat). The figure is originated from N Hashimoto (4), which is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/). EDA, esophagoduodenal anastomosis.

A total gastrectomy was performed and reconstruction was performed with an esophageal-duodenal anastomosis to create a model in which duodenal content refluxed into the esophagus. This procedure is the same as previously published (4).

Control rats (Control) (n=10); the sham operation, single laparotomy

Rats were sacrificed at 40^th week after surgery under general anesthesia. The middle and lower esophageal tissues were collected, half were frozen at −80 °C and used for the measurement of oxidative stress, and the other half were fixed with 10% formalin and stained with HE and COX2. We measured bile acids in the common bile duct and in esophageal lavage fluid.

Biochemical assays of esophageal mucosa

Esophageal tissue frozen at −80 °C was used for tissue malondialdehyde (MDA) (µmol/L), glutathione (GSH) (mg/dL) and superoxide dismutase (SOD) (µmol/L). MDA was determined according to the method of Buege and Aust. GSH level was measured according to Saville’s method. SOD activity was determined by the method reported by Laihia.

Immunohistochemistry

Localisation of COX2 protein was determined by immunohistochemical staining using specific antibodies. The DAKO EnVision system (Dako Cytomation Japan CO. Ltd., Kyoto, Japan) was used with autoclave acceleration. Finally, the localization of COX2 was visualized with diaminobenzidine tetrahydrochloride.

Measurement of bile acid in the esophageal lumen and the common bile duct

The collected esophagus was rinsed with 0.5 cc of saline, the solution was centrifuged at 1,500 rpm at 4 °C for 5 min, and the supernatant was measured for bile.

Moreover, bile was collected by inserting a thin tube into the common bile duct.

We measured bile acid concentrations with the ENZa BILE kit (Daiichi Pure Chemicals, Tokyo).

Statistical analysis

Data are expressed as mean ± standard deviation (SD) of each group. Student’s t-test was used for statistical analysis. P<0.05 was considered statistically significant.

Results

Macroscopic findings

In the lower and middle esophagus in EDA rat, thickening and shortening of the wall were observed, and a small polypoid mass was present.

Microscopic finding (Figure 2)

Figure 2 Microscopic findings in EDA rat (HE, ×200). EDA, esophagoduodenal anastomosis; SCC, squamous cell carcinoma; ADC, adenocarcinoma.

In EDA rat, 40 weeks after surgery, columnar-lined esophagus (CLE) was observed in 40%, SCC was observed in 40%, and adenocarcinoma (ADC) was observed in 30%.

Esophageal mucosal MDA (µmol/L), GSH (mg/dL) and SOD (µmol/L) activities (Figure 3)

Figure 3 MDA, SOD, GSH levels in esophageal tissues of EDA rat and Control rat. MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; EDA, esophagoduodenal anastomosis.

Mucosal MDA levels of EDA (69±4) were significantly higher compared to normal controls (52±11). GSH (267±50) and SOD levels (660±24) were significantly reduced in the EDA rat compared to the normal control rat (380±30, 822±104).

Immunohistochemistry of COX2 (Figure 4) (4)

Figure 4 COX2 staining of SCC and ADC in EDA rat (immunohistochemistry, ×200). The figure is originated from N Hashimoto (4), which is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/). COX2, cyclooxygenase 2; SCC, squamous cell carcinoma; ADC, adenocarcinoma; EDA, esophagoduodenal anastomosis.

SCC and ADC were strongly stained with COX2 protein in EDA rat. COX2 staining was strong in dysplastic and cancerous esophageal mucosa obtained from EDA rat (4).

However, COX2 staining was not observed in the esophageal mucosa from the control rat.

Composition and measurement of bile collected from the common bile duct (Table 1)

Total bile acid in the esophageal lavage: EDA rat (175±50 µmol/L) was significantly higher compared to the control rat (35±5 µmol/L).

Total bile acids and bile acid components collected from the common bile duct: total bile acids of common bile duct in EDA rat was significantly higher than that of control rat. Moreover, from the point of bile acid composition in common bile duct, taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA) in EDA rat were significantly higher than those in control rat.

Discussion

Most cases of esophageal cancer are occupied by SCC or ADC. Forty years ago, 75% of esophageal cancer in the United States was SCC and the remaining 25% was ADC. Among white men, ADC frequency has increased sharply since mid-1970. And ADC now accounts for over 80% of esophageal cancer cases (5). The rapid increase in the frequency of ADC in the western world is due to the increased frequency of GERD and obesity.

Gastroesophageal reflux disease (GERD) and Barrett’s esophagus are major risk factors for ADC, and the risk of developing ADC increases with the frequency and duration of reflux symptoms. We believe that frequent reflux of gastroduodenal fluid containing acids, pepsin, and bile induces Barrett’s esophagus.

Some clinical reviews have highlighted the significance of chronic duodenal gastroesophageal reflux in the development of Barrett’s esophagus (6). In addition, chronic esophageal reflux of duodenal contents in our EDA model caused CLE, SCC and ADC.

Bile acids are known to promote gastrointestinal cancer growth, but the underlying mechanism is unknown. Recently, there have been many reports on the significance of bile acids in carcinogenesis.

Bile acids stimulate cell signaling effects including c-myc, COX2 and nuclear factor (NF)-κβ, and therefore, bile acids may be involved in carcinogenicity (7). Nehra et al. (8) collected and analyzed bile acids in the esophagus from patients without GERD and with GERD. They reported that TCA and TDCA were significantly higher concentrations in the reflux fluid of patients with erosive stenotic esophagitis. Chen et al. (9) reported that a high-fat diet increased the proportion of taurine conjugates, and chronic exposure of TCA to the esophagus caused tumor progression in a rat reflux model. Hong et al. (10) reported that TDCA significantly increased cell proliferation in EA cells. Piessen et al. (11) showed that TCA, TDCA, and TCDA were potent activators of MUC 4 expression. MUC4 (mucin 4) is a membrane-bound mucin that is overexpressed in the early stages of esophageal carcinogenesis and is involved in tumor progression. Zhang et al. (12) reported that TCA, TCDCA and TDCA individually, and also in a mixture, induced apoptosis of cultured human normal esophageal mucosal epithelial cells. These bile acids are involved in esophageal carcinogenesis. In our rat model, analysis of bile acids in the common bile duct of total gastrectomized rats also showed that TCA, TDCA and TCDCA and TUDCA were significantly higher than those in control rat. We suggest that TCA, TDCA, TCDCA and TUDCA play an important role of esophageal carcinogenesis. In contrast, Nishioka et al. (13) reported that chenodeoxycholic acid (CDCA) stimulates proliferation of esophageal squamous cell carcinoma (ESCC) cells. These results suggest that the effect of bile acids on cell proliferation varies with bile acid type and cell line studies. Shirvani et al. (14) suggest that bile acids stimulate COX2 expression. These phenomena are associated with chronic inflammation and proliferation in cancer and Barrett’s esophagus.

We confirmed that COX2 was strongly stained in cancer and atypical mucosa obtained from rats with esophageal duodenal anastomosis. In contrast, COX2 was not stained in esophageal mucosa from the control rat. Moreover, oxidative damage has been proposed as a possible mechanism for human GERD and possibly also CLE. Barrett’s esophagus is a complication of GERD. This specialized intestinal metaplasia is considered a premalignant condition of the esophageal carcinoma that is rapidly increasing in incidence. For the development of carcinoma, oxidative stress has been suggested to be a driving force. Furthermore, ROS can activate a number of cancer-associated signaling pathways such as PI3K/Akt, ERK1/2, and NF-κB (15). In EDA rats, MDA in the esophageal tissue was higher than that in control rat, and SOD and GSH in the esophageal tissue were much lower than those in control rat. In our study, lipid peroxidation in the esophageal epithelium was significantly higher in the EDA rat than that of control rat. These results indicate a possible mechanism by which bile acids (TCA, TDCA, TCDCA, TUDCA) dramatically increase intracellular ROS levels and subsequently induce COX2 in a rat model of duodenal esophageal reflux (Figure 5). The elucidation of the detailed mechanism by which bile acids induce COX2 may facilitate the development of chemoprevention strategies to reduce the risk of carcinogenesis in gastroesophageal tracts exposed to bile acids.

Figure 5 Mechanism of bile acid reflux into the esophagus causing esophageal carcinogenesis in EDA rat. COX2, cyclooxygenase 2; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; EDA, esophagoduodenal anastomosis.

Conclusions

Bile reflux (TCA, TDCA, TCDCA, TUDCA) into the esophagus causes oxidative stress, subsequently induces COX2, and induces esophageal carcinogenesis.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The author has completed the ARRIVE reporting checklist. Available at http://dx.doi.org/10.21037/aoe-20-47

Data Sharing Statement: Available at http://dx.doi.org/10.21037/aoe-20-47

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/aoe-20-47). NH serves as an unpaid editorial board member of Annals of Esophagus from Apr. 2019 to Mar. 2021. The author has no other conflicts of interest to declare.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study complies with the ethical regulation of the use of laboratory animals and all experiments follow the animal experiment guideline in the Kindai University. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Kindai University (KAME-22-02).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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