I Received July 8, 1985; accepted December 23, 1985.

Transcription

1 Effects of Phenobarbital and Secondary Bile Acids on Liver, Gallbladder, and Pancreas Carcinogenesis Initiated by N-Nitrosobis(2-hydroxypropyl)amine in Hamsters 1,2 Takao Makino,3 Takeshi Obara,3 Hitoshi Ura,3 Tetsuo Kinugasa,3 Hironori Kobayashi,3 Seiichi Takahashi,3 and Yoichi KonishP.4 ABSTRACT-The effects of dietary administration of phenobarbital [(PB) CAS: ] or the secondary bile acids, deoxycholic acid [(DCA) CAS: ] and lithocholic acid [(LCA) CAS: ]. on tumorigenesis in the liver, gallbladder, and pancreas were investigated in male Syrian golden hamsters after carcinogenic initiation by N-nitrosobis(2-hydroxypropyl)amine [(BHP) CAS: ]. BHP [500 mg/kg (body wt)] was injected sc once weekly for 5 weeks. The animals were then maintained on a basal diet or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. DCA enhanced the development of cholangiocarcinomas without influencing that of hepatocellular lesions. PB promoted the induction of hepatocellular carcinomas but not that of cholangiocarcinomas. LCA was without effect on the induction of either hepatocellular carcinomas or cholangiocarcinomas. DCA at a dose of 0.5% enhanced the induction of polyps in the gallbladder. Both DCA, at a dose of 0.1%, and LCA significantly enhanced the induction of pancreas carcinomas. PB had no effect on the induction of polyps in the gallbladder or of pancreas carcinomas. These data document that different tumors may be differentially promoted following initiation with a common carcinogen.-jnci 1986; 76: Since the development of the concepts of initiation and promotion as applied to skin carcinogenesis (1), it has generally been accepted that these two qualitatively different stages also occur with neoplastic processes in other organs (2-4). The identification of intrinsic and environmental promoters in various experimental systems is recognized as an important research goal in chemical carcinogenesis. PB is a well-known promoter of hepatocarcinogenesis in rats (4-6) and mice (7). Bile acids promote colon (8-12), liver (13-15), and stomach (16, 17) carcinogenesis in rats. The potential promoting activities of PB on hepatocarcinogenesis in hamsters and that of the secondary bile acids, DCA and LCA, on gallbladder and pancreas carcinogenesis have not been considered previously. BHP is a broad-spectrum carcinogen first synthesized by Kruger et al. (18). Numerous studies of this compound have shown it to produce neoplasms in the pancreas, liver, and gallbladder of hamsters (19). In the present study, the influences of PB, DCA, and LCA treatment on the development of BHP-initiated lesions in the pancreas, liver, and gallbladder of hamsters were investigated. MATERIALS AND METHODS Animals.-A total of 163 male Syrian golden hamsters (Nippon Institute for Biological Science, Tokyo, japan), weighing approximately g each and 5-6 weeks old at the commencement of the experiment, were used. The hamsters were housed 5 per plastic cage in an airconditioned room at 24 C and 60% humidity, kept under a daily cycle of alternating periods of 12 hours of light and 12 hours of darkness, and given food and water ad libitum. All animals were fasted for 18 hours before being sacrificed under ether anesthesia. Chemicals and diets.-bhp (CAS: ) and DCA (CAS: ) were purchased from Nakarai Chemicals, Ltd., Kyoto, japan; LCA (CAS: ), from Sigma Chemical Co., St. Louis, MO; and PB (CAS: ), from Iwaki Seiyaku Co., Tokyo, japan. Commercial natural ingredient powder diet, Oriental MF from Oriental Yeast Co., Ltd., Tokyo, japan, was used as the basal diet. The diets containing either 0.05% PB, 0.1 % DCA, 0.5% DCA, or 0.5% LCA were prepared by mixing basal diet with the respective chemicals. The doses of DCA and LCA chosen for this study were determined after a subacute toxicity test for 10 weeks in hamsters according to the doses in previous reports (13, 20, 21). The dose of PB was the same as that demonstrating carcinogenic promoting potential previously in rats (22). Experimental protocol.-hamsters were divided into 10 groups according to the treatments as shown in textfigure 1. Groups 1-5 were the controls receiving sc injections of 0.9% NaCI once a week for 5 weeks, followed by a basal diet or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. Groups 6-10 received sc injections of 500 mg BHP Ikg (body wt) once a week for 5 weeks, followed by a basal diet alone or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. Body weight and food intake were measured weekly. All surviving hamsters were sac- ABBREVIATIONS USED: BHP = N nitl;osobis(2 hydroxypropyl)amine; DCA = deoxycholic acid; H &: E = hematoxylin and eosin; LeA = lithocholic acid; PB = phenobarbital. I Received July 8, 1985; accepted December 23, Supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture (No's and ) and by grants from the Ministry of Health and Welfare for Comprehensive 10-Year Strategy for Cancer Control, Japan. 3 Department of Oncological Pathology, Cancer Center, Nara Medical College, 840 Shijo-cho, Kashihara, 634 Nara, Japan. 4 Address reprint requests to Dr. Konishi. 967 jnci, VOL. 75, NO.5, MAY 1985

2 968 Makino, Obara, Ura, et al. G~ nn i~=~~~9=9==9= ~~ajo~o-o~.========~7~ ~ 0 > 0 I:::::::::::::::::::::::::::::::::::::::::;:;:;:;:;:;:;:;:;:;:;:;:::;:;:;:Wt::;:;:;:;:;:~,.. ~ ~ 0 0 0, ~JIr1r1 ~~uo >o~~ 5 L ~I::~~====~mmrnmmrnmm==--- L J. 6 7 _, t t Li t t! 1IIIIIIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIY.dIlIIIllIII ~ hc:."j t ~.~J.~J.~J. ~,, -:J.~I;lli:::::::~:::::::~:::::::m::::::~. ::::::~:::::::m:::::::~::::::~:::::;:m':':':'~:':"'''li'i';'i';'i'a,... r'... 8 Ii "h:rrt1 9 Lit t! ttttt~~ 10 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIY~ b ; ~ 1! ~!r---j!, Period (weeks) TEXT FIGURE I.-Experimental protocol. ~ = 0.9% NaCI sc; = BHP, 500 mg/kg (~wt) sc; 0= basal diet; tm:i)= diet con taining 0.05% PB; 1:Lj= diet containing 0.1% DCA; ~ = diet containing 0.5% DCA; DIDID= diet containing 0.5% LCA. rificed 35 weeks after the beginning of the experiment. The initial and effective numbers of hamsters are given in table 1. Histopathologic observations.-for histologic examination, liver sections were taken from macroscopically Group No. Treatment detectable nodules and each lobe and fixed in 10% buffered Formalin. To make sections from the gallbladder, we injected 10% buffered Formalin into the lumen and we cut longitudinally after fixation the gallbladder attached to the liver. The three splenic, gastric, and duodenal lobes of the pancreas were dissected out along with the common pancreatic duct and likewise fixed in 10% buffered Formalin. All tissues were embedded in paraffin, and sections were cut at 4-6 p,m in thickness and stained routinely with H & E. Periodic acid-schiff and alcian blue stains were used when appropriate. In the histopathologic examination, liver lesions were diagnosed according to the criteria described by Squire and Levitt (23) and according to the descriptions given by the Institute of Laboratory Animal Resources (24) for hyperplastic nodules, bile duct lesions, and carcinomas. Preneoplastic and neoplastic lesions in the pancreas were diagnosed according to the criteria described by Pour and Wilson (25) and Scarpelli et al. (26). Statistical differences in the incidence of the liver, gallbladder, and pancreas tumors were determined by Fisher's exact probability test (27). Other data were evaluated for significance with the use of Student's t-test (27). RESULTS TABLE 1.-Treatment, body weight, and organ weights of hamsters a No. of hamsters Initial Effective b Body wt, g Initial 1 Control ± % PB ±4" 3 0.1% DCA ± % DCA ± % LCA ±9 d 6 BHP ±9 7 BHP-0.05% PB ±11! 8 BHP-O.l% DCA ±5 9 BHP-0.5% DCA ±7 10 BHP-0.5% LCA ±6 h Final 164±12 162±20 168±14 143±l1 c 160±16 145±11 135±18 g 129±11! 107±14! 131±19 j The number of hamsters and their body weights, food intake, and liver and pancreas weights are given in table I. The effective number of hamsters was based on those accessible to histologic examination and excluded all animals that died during the first 18 weeks of the experiment. No tumors were found in any of these animals. Whenever sudden weight loss or obvious morbidity was noted after 18 weeks, the hamsters affected were sacrificed for histopathologic examination. A de- Average daily Liver wt Pancreas wt food intake, Percent of Percent of gjday g g body wt body wt 6.3±1.6 6,44± ± ± ± ±1.0 c 6.95± ± ±0.10d 0.37±0.07 c 6.4± ±0.81c 4.57±0.24c 0.80±0.13 0,48± ±0.7c 6.69± ±0.32c 0.59±0.12d 0,41±0.07 e 6.3± ±1.00 d 3.35±0.31c 0.79± ± ± ± ± ± ± ± ± ± ±0.17 h 0.46±o.o8 5.4± ± ±1.34Y 0.93± ± ±0.gf i 6.99± ± ± ± ±0.gf 9.21± ± ± ±0.21 j a Values are expressed as means ± SD. b Based on histologic examination. C Significantly different from group t, P<.005. d Significantly different from group 1, P<.01. e Significantly different from group 1, P<.025.! Significantly different from group 6, P<.005. g Significantly different from group 6, P<.05. h Significantly different from group 6, P<.Ol., Significantly different from group 8, P<.005. J Significantly different from group 6, P<.025.

3 PB, DCA, and LCA on BHP Carcinogenesis 969 creased final body weight was observed in animals from group 4 when compared with group I and in animals from groups 7-10 when compared with group 6. The average daily food intake per animal throughout the experimental period ranged from 4.6 to 6.4 g. The food intake decreased in animals from groups 2 and 4. It also decreased in animals from group 9 with respect to groups 6 and 8. Liver weight expressed as percent of body weight was elevated in animals from groups 3 and 4 and was decreased in animals from group 5 when compared with group l. It also increased in animals from group 8 when compared with group 6. Pancreas weight expressed as percent of body weight decreased in animals from groups 2 and 4 when compared with group I and increased in animals from group 10 when compared with group 6. Incidence of Hepatic Lesions Incidences of hepatocellular and bile duct lesions in the livers of hamsters treated with BHP followed by a basal diet or diets containing 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LeA are shown in table 2. Hyperplastic nodules developed in 100% of animals from groups Hepatocellular carcinomas developed in 13 of 24 (54%) animals from group 7, which was a statistically higher incidence than that of group 6 (P<'02). Bile duct proliferation was divided into four categories based on extent. It was observed to various degrees in animals from groups 6-10, and no statistically significant differences were evident among the groups. Cholangiocarcinomas developed in 10 of 19 (53%) animals from group 8 and in 9 of 25 (36%) animals from group 9, these both being significantly higher incidences than the incidence in animals from group 6 (P<'006 and P<'05, respectively). Liver lesions other than those arising from hepatocellular and bile duct cell components were limited to hemangioendotheliomas observed in 2 animals from group 7 and in 3 from group 8. No hepatic lesions were observed in animals from groups 1-5. Incidence of Gallbladder Lesions The incidences of gallbladder lesions arising in the different groups are shown in table 2. Presenting as polyps developing in the lumen of the gallbladder, their incidence was highest in group 9 animals, the increase in this group when compared to carcinogen control group 6 being statistically significant (P<'05). No polyps developed in the gallbladder of hamsters from groups 1-5. Incidence of Pancreas Lesions The incidences of pancreas lesions in hamsters treated with BHP followed by diets containing 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA are summarized in table 3. The incidences of gross tumors and carcinomas diagnosed histologically in animals from group 8 and the incidence of carcinomas in animals from group 10 were statistically higher than those in animals from group 6. However, no significant intergroup alteration in the incidence of adenomas or ductal hyperplasias, divided according to location with the pancreatic duct system, was evident. Pathology of the Lesions of the Liver, Gallbladder, and Pancreas Grossly, the liver showed multiple white spots and tan or whitish-yellow tumors, which were clearly demarcated from the surrounding tissue (fig. I). While tan tumors (right specimen in fig. I) presented relatively smooth surfaces and were soft in consistency, whitishyellow tumors (left specimen in fig. I) characteristically possessed an irregular surface and were hard in consistency. Some whitish-yellow tumors showed invasion and metastasis to the diaphragm and peritoneum (fig. 2). Histologically, liver lesions were classified as hyperplastic nodules, hepatocellular carcinomas, bile duct proliferations, and cholangiocarcinomas. Hyperplastic nodules showed characteristic features as described pre- TABLE 2.-Incidence of hepatocellular and bile duct lesions in the liver and gallbladder lesions in hamsters treated with BHP followed by diets containing 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA No. of hepatic lesions (%) Effective Group Treatment h No. ~f Hyperplastic Hepatocellular Bile duct proliferation No. of gallbladder a No. Chol~ngio- lesions or polyps (%) ams ers nodules carcinoma carcinoma 1 Control (100) % PB (100) % DCA (100) % DCA (100) % LCA (100) BHP (100) 2 (13) 0 11 (73) 4 (27) 0 1 (7) 2 (13) 7 BHP-0.05% PB (100) 13 (54)b 0 22 (92) 1 (4) 1 (4) 3 (13) 3 (13) 8 BHP-O.l% DCA (100) 2 (11) 0 16 (84) 3 (16) 0 10 (53)C 3 (16) 9 BHP-0.5% DCA (100) 1 (4) 0 11 (44) 10 (40) 4 (16) 9 (36)d 11 (44)d 10 BHP-0.5% LCA (100) 3 (14) 0 11 (50) 10 (45) 1 (5) 3 (14) 7 (32) a -, not observed; +, slightly observed; ++, moderately observed; +++, strongly observed. b Significantly different from group 6, P<.02. C Significantly different from group 6, P<.006. d Significantly different from group 6, P<.05.

4 970 Makino, Obara, Ura, et al. Group No. TABLE 3.-Incidence of pancreatic lesions in hamsters treated with RHP followed by diet containing 0.05% PR, 0.1% DCA, 0.5% DCA, or 0.5% LCA Treatment Effective No. of hamsters No. of pancreatic lesions (%) Microscopic findings Gross Ductal hyperplasia tumors a Carcinomas Adenoma CD+CPD MPD ID 1 Control % PB % DCA % DCA % LCA BHP 15 4 (27) 5 (33) 8 (53) 2 (13) 1 (7) 4 (27) 7 BHP-0.05% PB 24 8 (33) 9 (38) 13 (54) 6 (25) 7 (29) 3 (13) 8 BHP-O.l% DCA (74)h 14 (74)C 10 (53) 7 (37) 1 (5) 1 (5) 9 BHP-0.5% DCA 25 5 (20) 7 (28) 15 (60) 6 (24) 3 (12) 4 (16) 10 BHP-0.5% LCA (59) 15 (68)d 12 (55) 9 (41) 6 (27) 2 (9) a CD=common duct; CPD=common pancreatic duct; MPD=main pancreatic duct, ID=interlobular and intralobular ducts at the terminal branches of the ductal tree. b Significantly different from group 6, P<.009. C Significantly different from group 6, P<.03. d Significantly different from group 6, P<.04. viously (23, 24). Hepatocellular carcinomas were well differentiated and grew in trabecular patterns. Their nuclei were hyperchromatic and irregular in size, and mitoses were evident (fig. 3). Bile duct proliferation is synonymous with cholangiofibrosis, adenofibrosis, bile duct adenomatosis, fibroadenoma, and intestinal cell metaplasia, as referred to earlier (24). The lesions showed various degrees of extension and were characterized by foci or areas of hyperbasophilic atypical ducts in a fibrous stroma (fig. 4). Cholangiocarcinomas were composed of irregular glandular structures actively invading the surrounding tissue and were lined by an atypical epithelium. They were characterized by mucus production and abundant stroma. Mitotic figures were frequent (fig. 5). Metastasis and invasion to the diaphragm (fig. 6) and peritoneum were observed. The gallbladder polyps showed pedunculated and tree-like configurations with connective tissue stalks covered by a single layer of cuboidal or columnar epithelium. The polarity of epithelial cells was preserved. Mitotic figures were occasionally seen (fig. 7). In the pancreas, macroscopically the tumors appeared whitish-yellow and were hard in consistency. Histologically, well-differentiated tubular (fig. 8), papillary, or cystic adenocarcinomas were observed. Adenomas presented mainly as tubular structures lacking cellular atypia or invasive character. Ductal hyperplasias were observed in areas of the common duct and common pancreatic ducts, the main pancreatic ducts, and the interlobular and intralobular ducts at the terminal branches of the ductal tree. DISCUSSION The data presented above indicate that different cell types initiated by the same carcinogen respond to different promoting regimens. Especially in the liver, BHP alone yields few hepatocellular carcinomas or cholangiocarcinomas. Administration of BHP followed by PB resulted in a significantly increased incidence of hepatocellular carcinomas but not cholangiocarcinomas. In contrast, administration of BHP followed by DCA increased the yield of cholangiocarcinomas without effect on the number of hepatocellular carcinomas. To our knowledge, this is the first such demonstration of the ability of different promoters to produce different cancers following initiation by a single carcinogen within a single organ. The weekly dose of BHP (500 mg/kg) used in the present experiment was the same as that given previously by Pour et al. (19). Given for life, the incidence of hepatomas with this dose was 95% and that of cholangiocarcinomas was 58% in male Syrian golden hamsters. The lower total dose (2,500 mg/kg) used here, however, was still sufficient to initiate but not to promote neoplasia in hepatocytes, bile duct epithelial cells, and pancreatic duct cells. PB is well known to promote spontaneous hepatocellular tumors (28-30) and hepatocarcinogenesis by a variety of chemical carcinogens in mice and rats (7,31-33). The significant enhancement of hepatocarcinogenesis by PB documented in the present report contrasts with the previous report that PB failed to promote the induction of hepatocellular carcinomas in hamsters treated with aflatoxin BI (34). Cameron et al. (13) and Tsuda et al. (14) reported that DCA promotes the induction of phenotypically altered foci in the liver of rats that show positive 'Y-GTP activity. Tomatis et al. (35) observed that the hamster differed from the other species, including rats, in demonstrating a greater tendency for bile duct epithelial cells to proliferate in response to chemical carcinogens. The present result indicating promoting activity of DCA for the

5 PB, DCA, and LCA on BHP CarCinogenesis 971 development of cholangiocarcinomas may reflect such a species sensitivity of biliary epithelial cells. The development of cholangiocarcinoma was accompanied with various degrees of bile duct proliferation in hamsters from groups This finding is in close agreement with the proposed development sequence of cholangiocarcinomas leading from bile duct proliferation to cholangiocarcinomas (36). The absence of a dose-response in the promotion of cholangiocarcinoma by DCA may relate to nonspecific' toxic effects or depressed food intake in the higher dose. Caloric restriction has been shown to inhibit carcinogenesis (37, 38). The absence of LCA's promoting activity for the induction of hepatocellular carcinomas and cholangiocarcinom as is in agreement with the reports (13, 14) that LCA does not promote hepatocarcinogenesis in rats. Although bile duct proliferation was observed in hamsters from group 10, as reported earlier for LCA (20, 21), no promoting activity of LCA was recognized. In the gallbladder, polyps but not carcinomas were found. This result may be due simply to the 35-week period of the experiment. In a longer experiment, carcinomas of the gallbladder were frequent in hamsters treated with N-nitrosobis(2-oxopropyl)amine (39). It has been reported that BHP- or BOP-induced pancreatic carcinogenesis is effected via the blood stream (40-43). Although bile reflux has been implicated in the etiology of human pancreatic cancer (44), cholecystoduodenostomy with choledochostomy or partial pancreaticocolostomy failed to inhibit pancreatic cancer induction in hamsters (40, 41, 43). When DCA and LCA are fed, they are also absorbed and enter into the enterohepatic circulation (45). If the present documented promoting activities of DCA and LCA for pancreatic carcinogenesis are dependent on a bile reflux mechanism, carcinomas would be expected to be located in the head of the pancreas where the common duct and common pancreatic duct are located. Ductal hyperplasias and carcinomas, however, were not preferentially located in the head but were randomly scattered throughout all parts of the pancreas. Therefore, it seems that excess DCA or LCA in the blood may be responsible for the promotion of pancreatic tumor development. Further experimentation is required to clarify this point. In conclusion, the present results document that different target cells can have specificity for different promoters. Within a single organ, promoting chemicals given after an initiating carcinogen determine the type of tumor developed. Intrinsic chemicals such as DCA and LCA may exert promoting activity when their levels are at excess. REFERENCES (1) BERENBLUM 1. 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7 PB, DCA, and LCA on BHP Carcinogenesis 973 FIGURE I.-Macroscopic view of cholangiocarcinomas in a hamster from group 6 (left) and of a hepatocellular carcinoma (arrow) in a hamster from group 7 (right). FIGURE 2.-Macroscopic view of cholangiocarcinoma and metastases to the diaphragm (arrows) in a hamster from group 8.

8 974 Makino, Obara, Ura, et al. FIGURE 3.-Hepatocellular carcinoma in a hamster from group 10, showing a trabecular morphologic pattern. H & E. X 100 FIGURE 4.-Histologic specimen of a bile duct proliferating in a hamster from group 9. H & E. X 50 FIGURE 5.-Histologic detail from a cholangiocarcinoma in a hamster from group 8. H & E. X 160 FIGURE 6.-Histologic detail of a metastatic cholangiocarcinomatous lesion in the diaphragm of a hamster from group 8. H & E. X 80

9 r;~ ~~w - PB, DCA, and LCA on BHP Carcinogenesis 975 FIGURE 7.-GaIlhladder polyp arising in a hamster from group 6. H & E. X 32 ~ t FIGURE S.-Histologic detail of an adenocarcinoma in the pancreas of a hamster from group 10. H & E. X 50 jnci, VOL. 76, NO.5, MAY 1986