Acceleration of spontaneous biliary carcinogenesis in hamsters by bilioenterostomy

Tomoo Kitajima1, Yoshitsugu Tajima, Sumihiro Matsuzaki, Tamotsu Kuroki, Kenzou Fukuda and Takashi Kanematsu

Department of Surgery II, Nagasaki University School of Medicine, Nagasaki, Japan and Goto Central Hospital, 205 Yoshikugi, Fukue 853-0031, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Biliary carcinomas can occur as a delayed complication of bilioenterostomy. The aim of this study was to determine whether bilioenterostomy influences biliary carcinogenesis in hamsters. Syrian hamsters were subjected to three different surgical procedures: simple laparotomy (SL), choledochoduodenostomy (CD) and choledochojejunostomy (CJ). They were given no carcinogens, and five to six hamsters from each group were killed every 20 weeks up to 120 weeks after surgery. Thirty-seven, 32 and 38 hamsters were sampled from the SL, CD and CJ groups, respectively. Cholangiocarcinomas developed in 5.4, 15.6 and 23.7% of hamsters in the SL, CD and CJ groups, respectively. The incidence of biliary carcinoma was significantly higher in the bilioenterostomy groups, especially CJ (P < 0.05), than in SL. The tumor latency period after surgery was 20–40 weeks shorter in the bilioenterostomy groups than in SL. Persistent cholangitis and bile stasis were frequent in the bilioenterostomy groups, and a significant correlation between cholangitis and biliary carcinogenesis was noted in the CD group. The proliferative cell nuclear antigen (PCNA) labeling index was higher in the biliary epithelium of the bilioenterostomy groups. In conclusion, persistent cholangitis after bilioenterostomy accelerates biliary carcinogenesis through activation of biliary epithelial cell kinetics.

Abbreviations: CD, choledochoduodenostomy; CJ, choledochojejunostomy; LI, labeling index; PCNA, proliferative cell nuclear antigen; SL, simple laparotomy; T-Bil, total bilirubin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Biliary carcinomas sometimes develop after surgical treatments such as complete excision of the extrahepatic bile duct followed by biliary reconstruction for congenital cystic dilatation of the common bile duct (1,2) and bilio-enteric anastomosis for iatrogenic bile duct injury (3), and this results in a poor prognosis. Moreover, extremely high incidences of secondary biliary carcinoma have been reported recently in patients who have undergone biliary-enteric drainage for benign disease (4), transduodenal sphincteroplasty (5) or endoscopic sphincterotomy (6). Biliary reconstruction, enteric internal drainage and sphincteroplasty induce reflux of intestinal juice into the biliary tract due to disuse of the papillary muscle of the ampulla of Vater. Reflux of intestinal contents into the biliary system may influence the development of biliary carcinogenesis; however, the etiology of the secondary biliary carcinoma is not clear.

Many investigators have attempted to induce carcinomas in the biliary tree of laboratory animals in order to clarify the mechanism of biliary carcinogenesis (79). However, almost all the studies used chemicals in an attempt to induce biliary carcinomas, and there have been few reports of spontaneous biliary carcinoma.

Reconstruction of the biliary system is a common surgical technique that is widely used in the field of hepatobiliary pancreatic surgery. The present study was designed to clarify whether bilioenterostomy plays a role in biliary carcinogenesis in hamsters without any loading of chemical carcinogens. The Syrian golden hamster was used because the anatomical structure of its pancreaticobiliary ductal system and the bile acid composition and pancreatic juice components in this species are similar to those of humans (1012).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Seven-week-old female Syrian golden hamsters (SLC, Shizuoka, Japan) were housed, one per plastic cage, on sawdust bedding. They were kept at 24 ± 2°C and 50 ± 20% humidity with 12 h alternate light and dark, fed a CE-2 pelleted diet (Clea Japan, Tokyo, Japan) and provided with drinking water ad libitum. The animals were checked daily and weighed weekly during the experiment. All experiments were conducted according to the Guidelines for Animal Experimentation of Nagasaki University.

Surgical techniques
We used two types of bilioenterostomy that closely resemble those used in the clinical situation: choledochoduodenostomy (CD) and choledochojejunostomy (CJ). The hamsters were anesthetized with sodium pentobarbital (50 mg/kg body wt) and an upper-abdominal, midline incision was made. The distal end of the common bile duct was excised and the gallbladder was removed, then CD was performed on the duodenal wall ~10 mm distal to the pyloric ring of the stomach. CJ was achieved using ~4 cm of the jejunum for Roux-en-Y anastomosis (13,14). The control hamsters underwent simple laparotomy only (SL).

Morphological and physiological studies
To evaluate the morphological and physiological changes in the hepatobiliary system of hamsters after bilioenterostomy, five or six hamsters from each group were killed at 20-week intervals, from weeks 20 to 120 after surgery. At autopsy, the maximum diameter of the extrahepatic bile duct was measured. Bile in the biliary duct and blood samples from the vena cava were collected in ice-chilled tubes containing heparin, and then centrifuged (3000 r.p.m.) for 10 min. The serum levels of total bilirubin (T-Bil), glutamic–oxaloacetic transaminase (GOT), total bile acids (TBA) and amylase in the bile were then measured.

Pathological studies
The liver, biliary system and pancreas were removed en bloc. The specimens were fixed in 10% (v/v) neutral formalin, then cut into five blocks and embedded in paraffin. Histological sections were stained with hematoxylin and eosin (H and E), and then examined by a pathologist without prior knowledge of the treatments. The biliary neoplasms were diagnosed according to standard criteria (15).

Inflammatory changes
To evaluate the relationship between cholangitis and biliary carcinogenesis, the grade of cholangitis was scored in accordance with the infiltration of inflammatory cells: grade 0, no cholangitis; grade 1, mild invasion of inflammatory cells around the bile duct; grade 2, severe invasion of inflammatory cells around the bile duct; and grade 3, abscess formation in the liver.

DNA synthesis of the biliary epithelium
Proliferative cell nuclear antigen (PCNA) was detected in the specimens using anti-PCNA/horseradish peroxidase (Dako, Japan) after treatment with a household microwave oven. The proportion of labeled nuclei (labeling index, LI) was determined by counting the labeled nuclei in >1000 non-neoplastic epithelial cells of the intrahepatic and extrahepatic bile ducts.

Statistical analyses
The incidence of tumor development and appearance of cholangitis were analyzed statistically using the {chi}2 and Fisher’s exact tests. Student’s t and the Mann–Whitney tests were also used to compare body weight, diameter of the extrahepatic bile duct, number of tumors per animal and laboratory data on serum and bile juice in the treatment groups and controls.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Morphological and physiological changes in the hepatobiliary system
Table IGo summarizes the morphological and physiological changes in the hepatobiliary system of hamsters after bilioenterostomy. The total numbers of hamsters sampled were 37, 32 and 38 in the SL, CD and CJ groups, respectively. Almost all the hamsters in both the CD and CJ groups showed marked dilatation of the extrahepatic bile duct, and there were statistically significant differences in the average diameters of the extrahepatic bile ducts between the bilioenterostomized groups and the SL group. The serum levels of GOT, T-Bil and TBA did not differ among the three groups. The amylase activities in bile in the CD and CJ animals were high, although the difference was not significant.


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Table I. Morphological and physiological changes in the hepatobiliary system of hamsters after bilioenterostomy
 
Pathological studies
Spontaneous biliary neoplasms were observed in all three groups (Table IIGo). Representative cases of cholangioadenoma and cholangiocarcinoma are shown in Figure 1A and BGo. The occurrence rates of adenoma were 18.9, 28.1 and 23.7% in hamsters in the SL, CD and CJ groups, respectively, and there were no significant differences in the incidence of adenoma among the three groups. However, numerous biliary adenomas occurred in the bilioenterostomized animals, and the number of adenomas per tumor-bearing animal was much higher in the CD group than in the SL group (P < 0.01). Cholangiocarcinomas developed in 5.4% of hamsters in the SL group, compared with 15.6% in the CD group and 23.7% in the CJ group. The occurrence rate of cholangiocarcinoma in the CJ group was about four times higher than that in the SL group (P < 0.05). Carcinoma of the extrahepatic bile duct developed in two hamsters in the CD group and one hamster in the CJ group (Figure 1CGo), but no extrahepatic bile duct carcinoma occurred in the SL group. The latencies of the biliary tumors after bilioenterostomy are shown in Figure 2Go. Cholangioadenomas developed at postoperative week 40, 60 and 80 in the CJ, CD and SL groups, respectively. Cholangiocarcinomas occurred at postoperative week 60, 60 and 80 in the CJ, CD and SL groups, respectively.


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Table II. The incidence and number of tumors developed in hamsters after bilioenterostomy
 




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Fig. 1. (A) Intrahepatic cholangioadenoma observed in a hamster 59 weeks after CD (H and E, x200). (B) Intrahepatic cholangiocarcinoma observed in a hamster 83 weeks after CD (H and E, x200). (C) Extrahepatic bile duct carcinoma observed in a hamster 70 weeks after CJ (H and E, x100).

 


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Fig. 2. Tumor latency periods after bilioenterostomy in hamsters.

 
Cholangitis and biliary carcinogenesis
Cholangitis was observed in 54.1% of hamsters in the SL group, 81.2% in the CD group and 86.8% in the CJ group. Cholangitis of grade 2 or 3 was observed only in the bilioenterostomized animals, and the cholangitis scores in the CD and CJ groups were significantly higher than the score in the SL group. In the CD group, all hamsters except one with grade 2 or 3 cholangitis developed biliary carcinoma, and there was a significant correlation between the presence of cholangitis and biliary carcinogenesis (Figure 3Go). There was no such correlation in the SL and CJ groups.



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Fig. 3. Correlation between biliary carcinogenesis and cholangitis.

 
PCNA labeling indices
PCNA labeling indices (LI) of the biliary epithelia are summarized in Table IIIGo. The PCNA LIs in the enterostomized groups were higher than the index in the SL group and there was a significant increase in the LIs in the intrahepatic and extrahepatic bile duct in the CJ animals.


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Table III. PCNA labeling index in the bilary epithelium in hamsters after bilioenterostomy
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Spontaneous biliary tumors in Syrian hamsters have been reported by Pour et al. (16), and cholangiocarcinomas were observed in three of 118 female hamsters (2.5%) over a mean observation period of 71 weeks. In the present study, spontaneous cholangiocarcinomas occurred in two of 37 control hamsters (5.4%). The higher incidence of spontaneous cholangiocarcinomas compared with that observed by Pour et al. may be due to the longer observation period. However, in the bilioenterostomized hamsters, extremely high incidences of spontaneous cholangiocarcinomas were observed: occurrence rates of 15.6% in the CD group and 23.7% in the CJ group. Extrahepatic bile duct carcinomas were also observed in the enterostomized groups. Previously, we have reported the enhancement of biliary carcinogenesis in hamsters by cholecystoduodenostomy or cholecystoileostomy with use of a chemical carcinogen (7,8,14,17); however, the present study demonstrated the potent biliary carcinogenicity of bilioenterostomy alone, in hamsters, without the use of chemical carcinogens.

A clinical study has shown that bile duct cancer occurred in 55 of 1003 patients from biliary-enteric anastomosis between 11 and 19 years from operation (4) and in eight of 108 patients over a mean observation period of 17 years after sphincteroplasty (5). Spontaneous biliary neoplasms occurred 80 weeks after surgery in the SL group but bilioentrostomized hamsters developed biliary tumors at 40 weeks, which indicated that there was a difference in the latency period of tumor development between the groups. This evidence was also of interest because hamsters have a short life span of 100 to 150 weeks. We conclude, therefore, that the bilioenterostomy accelerated spontaneous biliary carcinogenesis in hamsters. Mechanical irritation by means of cholelithiasis (18,19), chronic intrahepatic cholangitis with hepatolithiasis (20), bile stasis and bacterial infection (21) have been thought to be causal factors in the development of biliary carcinoma. And Tocchi et al. (4) reported that cholestasis and cholangitis detected in the clinical history of 72% of patients who developed cholangiocarcinoma after biliary-enteric anastomosis. In the present study, cholangitis and bile stasis were observed in the bilioenterostomized hamsters and there was a significant correlation between the development of biliary carcinoma and the presence of cholangitis in the CD group. No such correlation was noted in the CJ group, although the incidence of biliary carcinoma and the grade of cholangitis were higher in the CJ group than those in the CD group. In the CJ group, eight hamsters exhibited grade 3 cholangitis with a liver abscess but no cholangiocarcinoma. Thus, the cholangitis appeared to have an effect on biliary carcinogenesis by means of its ‘persistency’ rather than its ‘severity’. In bilioenterostomized hamsters, moreover, non-tumorous biliary epithelium in the intrahepatic and extrahepatic bile ducts contained higher than normal levels of PCNA, a 36 kDa intranuculear protein which is used in immunohistochemical studies of epithelial cell proliferation (22,23). PCNA expression in the biliary epithelium may indicate increased proliferative activity in the epithelial cells and aggressive behavior of biliary carcinomas (2426). In the bilioenterostomized hamsters, PCNA LIs were elevated both at the periphery and in the center of the intrahepatic bile duct and most of the biliary carcinomas originated from the peripheral ductules in the biliary tree. These findings indicated that bile stasis should accelerate the progression and persistency of the peripheral cholangitis, and lead to an elevation of cell kinetic activity of the biliary epithelium and, eventually, cholangiocarcinoma.

In conclusion, spontaneous biliary carcinomas develop with an extremely high incidence in hamsters after bilioenterostomy in association with persistent chronic cholangitis. Thus, a high-risk state for biliary carcinogenesis may be created in the biliary tree when the biliary inflammation is induced by bilioenterostomy.


    Notes
 
1 To whom correspondence should be addressed Email: tokitajima-gi{at}umin.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 22, 2002; revised October 3, 2002; accepted October 7, 2002.