Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999), pp. 79 ± 86
Effect of Pancreatic Juice Re¯ ux into Biliary Tract on N-Nitrosobis(2-oxopropyl)amine (BOP)-Induced Biliary Carcinogenesis in Syrian Hamsters YOSHIFUMI O GURA, MD, SHINSUKE MATSUDA, MD, MASANOBU USUI, MD, NORIKO HANAMURA, MD, and YOSHIFUMI KAWARADA, MD, FACS
To elucidate the possible role of pancre atic juice re ¯ ux into the biliary tract in promoting the deve lopme nt of biliary carcinom a, Syrian hamsters were subje cted to chole cystoduode nostomy and ligation of the distal end of the common duct and the n subcutane ously inje cted with N-nitrosobis( 2-oxopropyl) amine (BO P) (e xpe rime ntal group) . The incide nce s of gallbladde r carcinoma and extrahe patic bile duct carcinoma in the e xpe rime ntal group was signi® cantly highe r than in the sham -ope rate d group (P , 0.01, P , 0.05) . The prolife rating cell nucle ar antige n (PCNA) labe ling indice s of both regions gradually increased with time, and were signi® cantly highe r in the experimental group at weeks 9 and 16 than in the sham-ope rated group at the same time. Trypsin and phospholipase A 2 (PLA 2) activitie s in bile and tissue le vels of supe roxide dismutase (SOD) in the gallbladde r and extrahe patic bile ducts were highe r in the expe rimental group than in the sham-ope rate d group. The se ® ndings sugge st that the carcinoge nic e ffe ct of BO P was enhance d in biliary epithe lium that had prolife rated in re sponse to and/or had be e n injure d by activate d pancre atic e nzyme s re¯ uxing into the biliary tract and then incre ase d fre e radical activity, le ading to a high fre que ncy of carcinom a deve lopme nt in the biliary tract. KEY WORDS: biliary cancer; trypsin; phospholipase A 2 ; supe roxide dismutase.
Conge nital biliary dilatation (CBD), so-calle d ª choledochal cystº or ª conge nital bile duct cyst,º is re lative ly common in Japan and othe r Asian countrie s. CBD is ge nerally associate d with pancre aticobiliary maljunction (PBMJ), a conge nital anomaly in which the union of the pancre atic and biliary ducts is locate d outside the duode nal wall (1), and the re is a high incide nce of carcinom a of the biliary tract in patie nts
Manuscript re ce ived February 18, 1998; acce pted June 22, 1998. From the First De partment of Surge ry, Mie University School of Medicine, Tsu Mie 514, Japan. Address for reprint requests: Dr. Yoshifumi Ogura, First De partme nt of Surgery, Mie Unive rsity School of Medicine, 2-174 E dobashi, Tsu Mie 514, Japan.
with CBD associate d with PBMJ (2). Re¯ ux of pancreatic juice into the biliary tract through the associated PBMJ is conside red to be a factor promoting the de velopme nt of carcinoma of the biliary tract in patie nts with CBD (3, 4), and the possibility of malignancy in CBD is also known to be enhance d by e nte ric internal drainage (5± 7). Howe ve r, the precise mechanism of carcinoge ne sis in CBD and PBMJ is still unknown. In the prese nt study, we inve stigate d the possible role of re ¯ ux of pancre atic juice into the biliary tract on BO P-induce d carcinoge ne sis in the biliary tract of Syrian hamsters, focusing on activate d pancre atic e nzyme s and fre e radicals.
79
Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999) 0163-2116/99/0100-0079$16.00/0 Ñ
1999 Plenum Publishing Corporation
OGURA ET AL
Fig 1. E xpe rime ntal protocol for the rapid-production model of biliary tract carcinogene sis induced by BO P in hamsters.
MATERIALS AND METHODS Anim als A total of 86 ® ve -we e k-old female Syrian golden hamsters (Shizuoka Laboratory Animal Center Co. Ltd., Shizuoka, Japan) we re housed three per cage and maintained under standard laboratory conditions in the Laboratory Animal Center for Biochemical Research, Mie Unive rsity School of Medicine. The animals we re give n a standard pellet diet and wate r ad libitum throughout the study. All e xpe riments were conducted in accordance with the Guidelines for Animal Expe rimentation of Mie Unive rsity.
Su rgical Tech niqu es The e xtrahe patic biliary tract of the hamster is composed of the gallbladder and extrahe patic bile duct, ie, common bile duct and common duct. Completed surgical procedures consisted of cholecystoduodenostomy and double ligation of the e xtrahe patic bile duct at the distal e nd of the common duct so that pancreatic juice would ¯ ow backward into the biliary tract. Following intraperitoneal ane sthesia with Ne mbutal (50 mg/kg of body weight), a midline upper abdominal incision was made, and the distal e nd of the common duct was doubly ligate d with 9-0 nylon. A 1-mmlong incision was made in both the gallbladder fundus and the duodenal wall approximately 10 mm distal to the pyloric ring, and cholecystoduodenostomy was performed by continuous suture with 9-0 nylon using microsurgical techniques. The sham-operated hamsters were subjected to simple laparotomy.
Carcin ogen esis Stu dy Carcinogen . The carcinogen used was N-nitrosobis(2oxopropyl)amine (BOP, Nakarai Chemical Co. Ltd. Kyoto) dissolved in physiological saline. Experim ental Protocol. The hamste rs in the expe rimental group (19 animals) we re subjecte d to cholecystoduodenostomy and double ligation of the distal end of the common duct and, starting one we e k afte r surgery, we re subcutaneously injecte d with BOP, 10 mg/kg body we ight, once a wee k for 10 consecutive wee ks. The 15 hamste rs in the sham-operate d group we re subjecte d to simple laparotomy, given the same doses of BOP, and observe d for the
80
same period as the animals in the e xperimental group. The ® ve hamsters in the normal control group we re not subjected to any surgical operations or injected with BO P, but were observe d for the same period as the animals in the other two groups. At 16 we e ks afte r the operation or the start of the e xperiment, surviving animals were killed with an overdose of Nembutal. Animals that died during the period of the expe riment we re e xcluded from the analysis. Imme diate ly afte r death, the extrahe patic bile ducts and gallbladder we re remove d en bloc togethe r with the liver, pancreas, and part of the duodenum, and ® xed in 10% neutral formalin (Figure 1). Histological Exam ination . The formalin-® xed specimens were cut into 10 ± 13 sections and embe dded in paraf® n. All of the sections we re stained with hematoxylin and eosin (H& E). The number of histologically veri® ed carcinomas of the biliary tract, ie, of the gallbladder and extrahe patic bile ducts, was counted, with particular atte ntion to the location and growth patte rn of the tumors induced. Carcinoma was diagnosed on the basis of disruption of the polarity of the e pithelial cells; evidence of an invasion, and cytoplasmic or nuclear atypia. Cell K inetic Stu dies. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was performed by the streptavidin± biotin-complex method (sABC kit, Nichirei, Tokyo). Tissue deparaf® nization was achieve d with xyle ne, and dehydration with a graded alcohol series. The sections we re imme rsed in 0.3% hydrogen peroxide in absolute methanol for 30 min at room te mpe rature and washed in phosphate-buffe red saline (PBS, pH 7.2). Following incubation with normal rabbit serum (Nichirei, Tokyo) for 10 min, the sections we re incubated with primary antibody for 12 hr in a humidi® e d chamber at room temperature. The primary antibody used was a 1:100 dilution of anti-PCNA (PC-10, Dako, Glostrup, De nmark). Afte r incubation, the sections we re washed with PBS three times and tre ate d with biotinylated goat anti-mouse IgG and peroxidase-conjugate d streptavidin for 30 min. They we re the n reacte d with 0.02% diaminobenzidine te trahydrochloride containing 0.005% hydrogen peroxide for 3 min, counte rstained with hematoxylin, dehydrate d, cleare d, and mounted. The PCNA labeling index (LI) was dete rmined by selecting the most PCNA-positive are a from seve ral highpower microscopic ® elds ( 3 400) and counting labeled and Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999)
BILIARY CARCINOGENESIS BY BOP T ABLE 1. I NCIDENCE OF G ALLBLADDER AND E XTRAHEPATIC B ILE D UCT C ARCINOMA IN H AMSTER E XPERIMENTS
G roup E xperime ntal Sham-ope rated Normal control
Ham sters with carcinom a [N (% )] Ham sters (N) G allbladder Extrahepatic bile ducts 19 15 5
15 (79) * 1 (7) 0
7 (37) ² 1 (7) 0
* Signi® cantly different from the sham-operate d group (P , ² Signi® cantly different from the sham-operate d group (P ,
0.01). 0.05).
unlabeled cells until reaching a total of 500 cells in the area selected. The LI was the n calculated as the ratio of PCNAlabeled cells to the total number of cells counted, and e xpressed as a percentage . Biological Stu dies. The animals we re ane sthesized, and the contents of the extrahe patic bile duct we re collected and stored at 2 80 °C until samples were assaye d for trypsin and phospholipase A 2 (PLA 2 ) activitie s. Trypsin activity was measured by double-antibody radioimmunoassay (8), and PLA 2 activity was analyzed by measuring the amount of radiolabe le d fatty acid re le ase d from the substrate , 1-stearoyl-2-[ 1- 1 4 C]arachidonyl phosphatidylcholine (PC) (Ame rsham, Japan), according to the me thod of Isaji (9). Afte r homoginizing the gallbladder and e xtrahepatic bile duct specimens with distilled wate r and centrifuging the m, the supernatants we re assayed for superoxide dismutase ( SO D) activity with 5,5 -dime thyl-1-p yroline -N-oxide (DMPO ; Labote c, Tokyo, Japan) by the spin-trap me thod of Hiramatsu et al (10) on an electron spin resonance spectrometer (JES-RE1X; Jeol, Tokyo, Japan). The specime ns obtained we re then homogenized in 2 ml of 0.1 M glycine± borate buffer (pH 8.45).
Morp h ological Typ es an d Micros cop ic Fin din gs of Biliar y Tract Carcin om as The carcinomas of the biliary tract were classi® e d into the following morphological type s: (1) tumors with papillary or nodular tumors that proje cted into the lume n and (2) supe r® cially spre ading tumors. Gross e xamination of the le sions in the experimental group reve aled that 17 (94% ) of the 18 carcinomas of the gallbladde r and 13 (93% ) of the 14 carcinom as of the extrahe patic bile ducts were type 1, ie, were papillary or nodular type in form and proje cted into the lumen (Figure 2a) . Papillary ade nocarcinoma accounte d for 14 (78% ) of the 18 carcinomas of the gallbladde r and 8 (57% ) of the 14 carcinomas of the e xtrahe patic bile ducts in the e xpe rimental group (Figure 2b) . Tubular ade nocarcinom a was found in 4 (22% ) of the carcinomas of the gallbladde r and 6 (43% ) of the carcinomas of the e xtrahe patic bile ducts in the experimental group, but none were found in the control group (Table 2). PCNA LI The LIs of the gallbladde r and the e xtrahe patic bile ducts in the expe rime ntal group gradually incre ase d with time and were signi® cantly highe r in the e xpe rime ntal group than in the normal controls at wee ks 6, 9, and 16 afte r the ope ration. The LIs were signi® cantly highe r in the e xpe rime ntal group at wee ks 9 and 16 than in the sham-ope rate d group at same wee k (Table 3). Tryp sin an d PLA2 Activities in Bile
Statistical Analys is The incidence of tumor induction was statistically analyzed by the chi-square te st. Student’s t te st was used to compare the number of tumors per animal. PCNA LIs, trypsin PLA 2 and SOD activities. A signi® cant difference was de® ned as P , 0.05.
Trypsin and PLA 2 activitie s in bile are shown in Table 4. Both activitie s in bile were signi® cantly highe r in the expe rimental group at wee ks 3, 6, 9, and 16 after the ope ration than in the normal controls, and both activitie s were signi® cantly highe r in the e xpe rime ntal group at weeks 3, 6, 9, and 16 than in the sham-ope rate d group at the same wee k (Table 4).
RESULTS Inciden ce of Gallb lad der an d Extrah epatic Bile Duct Carcin om a in Ham sters Treated with BOP The incide nces of carcinoma of the gallbladde r and of carcinoma of the extrahe patic bile ducts induce d in e ach group are shown in Table 1. The differe nce s be twe e n the e xpe rime ntal group and the sham ope rated group in incide nces in both re gions were statistically signi® cant (carcinom a of the gallbladde r: P , 0.01, carcinoma of the e xtrahe patic bile ducts: P , 0.05) (Table 1). Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999)
SOD Activity in Gallbladder an d Extrah epatic Bile Duct Tissue SO D activity in the gallbladde r and e xtrahe patic bile duct tissue was signi® cantly highe r in the expe rime ntal group at weeks 3, 6, and 16 afte r ope ration than in the normal group. SOD activity in gallbladde r tissue was signi® cantly highe r in the experimental group at wee ks 3, 6, and 16 afte r the ope ration than in the sham-ope rate d group at the same weeks, and SO D activity in extrahe patic bile duct tissue was signi® cantly highe r in the expe rimental group at wee ks
81
OGURA ET AL
Fig 2. (a) A papillary projection type carcinoma in the gallbladder of a hamster in the e xperime ntal group (H& E staining, 3 60). (b) Highe r magni® cation of the tumor in Figure 2a, re vealing papillary ade nocarcinoma (H& E staining, 3 100) .
3, 6, 9, and 16 after the ope ration than in the shamope rated group at same wee ks (Table 5). DISCUSSION The mechanism of carcinoge nesis in chole dochal cyst and pancre aticobiliary malunion still remains unknown. Howe ver, some bile acid fractions (lithocholic and de oxycholic acid) (2) and re gurgitate d pancre atic e nzymes in bile [activate d trypsin (11) , elastase I, and PLA 2 (12) ] are toxic to the e pithe lium and may promote carcinoma in the pre se nce of infection, in¯ ammation, bile stasis, decrease d trypsin inhibitor le ve ls, and e nte rokinase . Many atte mpts have bee n made to induce carcinoma in the biliary tract of laboratory animals (13± 19) as a means of clarifying the characte ristics of human carcinoma of the biliary tract, but the re sults have bee n unsatisfactory. Pour et al (20, 21) and Kamano et al (22) reporte d a ne oplastic re sponse of the pancre aticobiliary syste m to subcutane ous adm inistration of BOP in hamste rs. They stated that the re was no e vide nce of neoplastic growth in the common bile duct or he patic ducts, but that a slight te nde ncy toward malignancy and neoplasm formation was ob-
82
se rved in the common duct. BOP is also well re cognized as a pote nt pancre atic carcinoge n in the hamster. In 1994, Tajima et al (23) reporte d a ne w model with a high incide nce of tumor de velopme nt in the e xtrahe patic bile ducts and gallbladde r in the hamster. They pe rforme d chole cystoduode nostomy and disse cted the e xtrahe patic bile duct at the distal e nd of the common duct in hamsters so that pancre atic juice ¯ owe d backward into the biliary tract, and the n administe red BO P. To elucidate the role of the activitie s of various prote inase s and the contributions of fre e radical activity as a result of pancre atic juice re ¯ ux into the biliary tract in promoting the de velopme nt of carcinoma of the biliary tract, we pre pare d the ham ste r mode l describe d by Tajima e t al. In this study, the incide nces of carcinoma of the gallbladde r and extrahe patic bile ducts in the experimental group were signi® cantly highe r than in the sham-ope rate d group. Most of the induce d carcinomas we re of the papillary or nodular type morpholo gically, a patte rn characte ristic of e arly carcinoma of the biliary tract in humans ( 24) , and the most com mon histolog ical type was papillar y ade nocarcinoma, accounting for Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999)
BILIARY CARCINOGENESIS BY BOP
Fig 2. Continued.
78% of the gallbladd e r carcinom as and 57% of the e xtrahe patic bile duct carcinomas in the e xpe rime ntal group. Cells in the DNA synthe sis phase of the cell cycle are most susce ptible to the tumorige nic effects of che mical carcinoge ns (25) , and DNA-synthe sizing ce lls can be ide nti® e d by imm unohistoche mical stainin g me thods using anti-PC NA monoclonal antibody ( 26, 27) . In this study, the LIs of the gallbladde r and the e xtrahe patic bile ducts graduall y incre ase d with time and we re signi® cantly highe r in T ABLE 2. M ORPHOLOGICAL T YPES
AND
the e xpe rime ntal group at we e ks 9 or 16 afte r ope ration than in the sham -ope rate d group at same time s. Fujita et al (28) re porte d that the gallbladde r e pithelium of patie nts with PBMJ exhibits incre ase d cell prolife ration, which promote s carcinoge nesis. The activitie s of various prote inase s are conside re d to be close ly conne cted with carcinoge nic proce sse s in which a tumor promotion proce ss is implicate d (29, 30) . It has bee n re porte d that trypsin functions associate d with cell division and the promotion proce ss
H ISTOLOGICAL FINDINGS
OF
C ARCINOMA
OF THE
Morphological types [N (% )] G roup Carcinoma of the gallbladder Experimental Sham-ope rated Normal control Carcinoma of the e xtrahepatic bile duct Experimental Sham-ope rated Normal control
B ILIARY TRACT Histological ® ndin gs [N (% )]
Ham sters (N)
Ham sters with Ca. (N)
Total no. of Ca.
Papillary an d nodular
Super® cial spread
Papillary adenocarcin om a
Tubular adenocarcin om a
19 15 5
15 1 0
18 1 0
17 (94) 1 (100) 0
1 (6) 0 0
14 (78) 1 (100) 0
4 (22) 0 0
19 15 5
7 1 0
14 2 0
13 (93) 1 (50) 0
1 (7) 1 (50) 0
8 (57) 2 (100) 0
6 (43) 0 0
Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999)
83
OGURA ET AL T ABLE 3. P ROLIFERATING C ELL NUCLEAR A NTIGEN (PCNA) LABELING I NDEXES OF H AMSTERS
IN
B ILIARY E PITHELIUM
PCNA labeling index (% ) G roup
Experim ental period (weeks)*
Ham sters killed (N)
3 6 9 16 3 6 9 16 16
5 5 5 5 5 5 5 5 5
Expe rimental group
Sham-operate d group
Normal control
G allbladder 4.0 12.5 31.3 69.1 3.4 4.4 5.0 11.8 3.3
6 6 6 6 6 6 6 6 6
Extrahepatic bile du cts
3.6 7.8a² 5.4b, d 19.5c, e 2.7 3.6 4.8 9.1 3.9
4.0 11.2 24.5 47.4 3.1 4.3 6.0 10.3 3.1
6
3.3 6.3f 8.6g, i 18.5h, j 2.1 3.2 4.1 7.5 2.7
6
6
6
6
6
6
6
6
* Interval be tween the operation and killing. ² a, b, c: signi® cantly diffe rent from the normal control (e ach P , 0.01) ; d: signi® cantly different from the sham-ope rated group at 9 wee ks (P , 0.01) ; e : signi® cantly different from the sham-operate d group at 16 we eks (P , 0.01) ; f, g, h: signi® cantly different from the normal control (P , 0.05, P , 0.01, P , 0.01) ; i: signi® cantly different from the sham-operated group at 9 wee ks (P , 0.01) ; j: signi® cantly different from the sham-ope rated group at 16 wee ks (P , 0.05) .
may be relate d to depre ssion of the genome by histone modi® cation, pe rmitting incre ase d transcription. Nakamura e t al (31) re porte d that trypsin incre ase d 3 incorporation of H-labe led thymidine into DNA in a chick embryo ® broblast culture system. PLA 2 conve rts lechithin to lysole cithin, which is very toxic to the mucosal barrie r (32) , and plays an important role because of its e ffe ct in breaking down the cell membrane and in association with the production of lipid pe roxide , which cause furthe r cell damage (33) . In this study, we found evide nce that trypsin and PLA 2 were highly activate d in the stagnant bile in the exT ABLE 4. A CTIVITIES
OF
pe rime ntal group. We the re fore conclude d that the forme r acte d to stimulate cell prolife ration and that the latte r cause d cell damage to the biliary e pithe lium. In this study, we asse sse d the contribution of fre e radicals, which play an important role in the carcinogenic mechanism (34, 35) . We measure d SO D activity in gallbladde r and e xtrahe patic bile duct tissue , since most of the active oxyge n gene rated in response to the administration of carcinoge nic substance s is inactivate d by the free radical scavenging system, including SO D, catalase , and GSH-Px in the lipids of the
P ANCREATIC E NZYMES IN B ILE G ROUPS
IN
E XPERIMENTAL
AND
SHAM -O PERATED
Activities of the pancreatic enzymes in bile G roup Expe rimental group
Sham-operate d group
Normal control
Experim ental period (weeks)*
Ham sters killed (N)
3 6 9 16 3 6 9 16 16
5 6 5 7 5 5 5 5 5
Trypsin (ng/m l) 491.9 523.1 467.8 591.7 163.3 201.8 198.5 208.0 195.0
6
6
6
6
6
6
6
6
6
170.3a, e² 124.9b, f 137.3c, g 148.2d, h 43.7 67.0 53.2 23.9 37.0
PLA 2 (units/m l) 25.8 26.2 29.4 30.2 8.9 9.5 11.2 10.7 7.9
6
6
6
6
6
6
6
6
6
7.9l, m 6.2j, n 9.3k, o 9.1l, p 2.5 4.7 8.9 4.0 3.6
* Interval be tween the operation and killing. ² a, b, c, d: signi® cantly differe nt from the normal control (e ach P , 0.01) ; e : signi® cantly different from the sham-ope rated group at 3 we eks (P , 0.01) ; f: signi® cantly different from the sham-operated group at 6 we eks (P , 0.01) ; g: signi® cantly differe nt from the sham-operate d group at 9 we eks (P , 0.01); h: signi® cantly different from the sham-ope rated group at 16 wee ks (P , 0.01) ; i, j, k, l: signi® cantly differe nt from the normal control (P , 0.05, P , 0.01, P , 0.01, P , 0.01) ; m: signi® cantly different from the sham-operate d group at 3 wee ks (P , 0.01) ; n: signi® cantly different from the sham-operate d group at 6 we eks (P , 0.01) ; o: signi® cantly different from the sham-ope rated group at 9 weeks (P , 0.05); p: signi® cantly different from the sham-operate d group at 16 we eks (P , 0.01) .
84
Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999)
BILIARY CARCINOGENESIS BY BOP T ABLE 5. SUPEROXIDE
DISMUTASE
(SOD) A CTIVITY IN G ALLBLADDER TISSUE
AND
E XTRAHEPATIC B ILE D UCT
SOD activity (units/m g protein) G roup Expe rimental group
Sham-operate d group
Normal control
Experim ental period (weeks)*
Ham sters killed (N)
3 6 9 16 3 6 9 16 16
5 6 6 8 5 6 7 8 5
G allbladder 26.5 29.3 29.1 36.3 16.1 17.3 18.1 18.7 16.6
6
6
6
6
6
6
6
6
6
3.9a, d² 9.8b, e 12.6 13.8c, f 2.7 3.7 4.2 4.2 5.4
Extrahepatic bile duct 17.9 17.2 23.2 26.9 8.4 7.2 7.4 9.3 7.5
6
6
6
6
6
6
6
6
6
7.2g, j 2.3h, k 15.3l 5.4l, m 4.5 2.0 1.9 3.8 2.7
* Interval be tween the operation and killing. ² a, b, c: signi® cantly different from the normal control (P , 0.05, P , 0.05, P , 0.01); d: signi® cantly differe nt from the sham-ope rated group at 3 wee ks (P , 0.01) ; e: signi® cantly diffe rent from the sham-ope rated group at 6 we eks (P , 0.05) ; f: signi® cantly differe nt from the sham-operated group at 16 we eks (P , 0.01); g, h, i: signi® cantly different from the control group (P , 0.05, P , 0.01, P , 0.01) ; j: signi® cantly differe nt from the sham-operated group at 3 wee ks (P , 0.05); k: signi® cantly different from the sham-operated group at 6 we eks (P , 0.01); l: signi® cantly different from the sham-ope rated group at 9 wee ks (P , 0.05) ; m: signi® cantly diffe rent from the sham-operate d group at 16 we eks (P , 0.01) .
cell membrane and cytoplasm before is re ache s DNA. An incre ase of SO D activity in the e xpe rime ntal group may have playe d a role in inactivating fre e radicals in the carcinoge nic proce ss. In conclusion, the results of this study indicate that the carcinoge nic e ffe ct of BO P was enhance d in biliary epithe lial cells that had prolife rate d in response to and/or had bee n injure d by activate d pancre atic e nzyme s ¯ owing backward into the biliary tract, and then incre ase d fre e radical activity, le ading to a high fre que ncy of carcinoma de velopme nt in the biliary tract. REFERENCES 1. The Japanese Study Group on Pancreaticobiliary Maljunction (JSPBM) . The Committee of JSPBM for Diagnostic Criteria: Diagnostic criteria of pancreaticobiliary maljunction. J Hepat Bil Pancr Surg 1:219 ± 221, 1994 2. Todani T, Watanabe Y, Uchiyama N, Moritomi Y, Maeba T: Choledochal cyst, pancre atobiliary malunion, and cance r. J Hepat Bil Pancr Surg 1:247± 251, 1994 3. Kinoshita H, Nagata E, Hirohashi K, Sakai K, Kobayashi Y: Carcinoma of the gallbladder with an anomalous conne ction be tween the chole dochus and the pancreatic duct; Report of 10 cases and re vie w of the literature in Japan. Cancer 54:762± 769, 1984 4. Loria LE, Yamamoto K, E to T, Tomioka T, Miyamoto T, Mochinaga N, Tsuchiya R: A case of a rare anomaly of the common bile duct associate d with an abnormal arrange ment of the pancre aticobiliary ductal union. Jpn J Surg 18:718 ± 724, 1988 5. Flanigan DP: Biliary cysts. Ann Surg 182:635± 643, 1975 6. Tsuchiya R, Harada N, Ito T, Furukawa M, Yoshihiro I, Kusano T, Uchimura M: Malignant tumors in chole dochal cysts. Ann Surg 186:22± 28, 1977 Digestive Diseases and Sciences, Vol. 44, No. 1 (January 1999)
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