Chronic endogenous hypercholecystokininemia promotes pancreatic ...

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Carcinogenesis vol.18 no.2 pp.315–320, 1997

Chronic endogenous hypercholecystokininemia promotes pancreatic carcinogenesis in the hamster

Ming Chu, Jens F.Rehfeld1 and Kurt Borch2 Department of Surgery, University Hospital of Linko¨ping, Linko¨ping, Sweden and 1Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark 2To

whom correspondence should be addressed

In order to examine the effect of cholecystokinin on spontaneous and induced pancreatic carcinogenesis in the hamster, two sets of experiments were carried out, one involving long-term hypercholecystokininemia and one involving cancer induction during hypercholecystokininemia. The effect of hypercholecystokininemia, induced by pancreaticobiliary diversion (PBD), was studied for 8 months. Neither PBD animals nor sham-operated controls developed premalignant or malignant pancreatic lesions. However, in the PBD group the mean pancreatic weight, total protein content and DNA content were increased by 30, 29 and 27% respectively. No such increases were found in PBD animals receiving a cholecystokinin-A receptor antagonist during the last 24 days of the experiment. In the cancer induction study, the effect of PBD on N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis was studied for 3 months. Putative premalignant pancreatic lesions were diagnosed in all PBD hamsters and in four of 15 sham-operated controls. Pancreatic ductular carcinoma in situ was only found in PBD animals. The [3H]thymidine labeling index of the pancreatic lesions was significantly higher in the PBD group than in the controls. No such increase was observed in PBD animals receiving a cholecystokinin-A receptor antagonist during the last 5 days of the experiment. It is concluded that chronic endogenous hypercholecystokininemia promotes early phase pancreatic carcinogenesis, but does not per se cause development of premalignant or malignant pancreatic lesions in the hamster. Introduction A trophic effect of exogenous and endogenous cholecystokinin (CCK*) on the exocrine pancreas in the rat is well documented (1–14). This effect is mediated by CCK-A receptors (14). Exogenous and endogenous CCK have also been shown to promote azaserine-induced pancreatic carcinogenesis, leading to acinar cell neoplasia in the rat (15–20). Studies on the effect of CCK in the hamster nitrosamine model, which leads to development of ductal pancreatic neoplasia (21), show conflicting results ranging from promotion (22–24), to no effect (25–28), to inhibition (25,29–32). Compared with acinar cell carcinogenesis in the rat, less is known about the possible hormonal mechanisms involved in neoplastic changes of ductal tissue induced by N-nitrosobis(2oxopropyl)amine (BOP) in the hamster. *Abbreviations: CCK, cholecystokinin; BOP, N-nitrosobis(2-oxopropyl) amine; PBD, pancreaticobiliary diversion. © Oxford University Press

In the hamster, as well as in the rat (5–8), pancreaticobiliary diversion (PBD) with transposition of the orifice of the pancreaticobiliary ducts distally in the small intestine induces endogenous hypercholecystokininemia with ensuing pancreatic hyperplasia and hypertrophy (33,34). This trophic effect is prevented by simultaneous administration of a specific CCKA receptor antagonist, such as L-364,718 (34). PBD thus seems to be a useful model for investigating the effect of long-term endogenous hypercholecystokininemia on pancreatic carcinogenesis without laborious and non-physiological exogenous CCK administration or special feeding. The purpose of the present study was to investigate the effect of PBD with chronic endogenous hypercholecystokininemia on spontaneous and BOP-induced pancreatic carcinogenesis in the hamster. Sham-operated animals and PBD animals treated with a CCK-A receptor antagonist served as controls. Materials and methods Animals This study was approved by the local animal welfare committee. Eighty eight 10-week-old male Syrian golden hamsters (Bantin and Kingman, North Humberside, UK) with a mean body weight (6 SD) of 80 6 7 g were used. The animals were kept at 20°C, 50% humidity on a light/dark cycle of 12/12 h. They had free access to standard hamster food pellets (Lactamin, Vadstena, Sweden) and tap water. Long-term hypercholecystokininemia study At 12 weeks of age, 35 hamsters were randomised to undergo PBD (9) by transposing, at most, 7 mm of the duodenum, including the pancreaticobiliary ducts and papilla, to the middle of the small intestine. Ten animals were sham operated with small intestinal transection (controls). Ketamine hydrochloride (Ketalar; Parke-Davis, Barcelona, Spain) and xylazin chloride (Rompun; Bayer, Malmo¨, Sweden) given i.p. were used for general anesthesia. The animals were fasted for 15 h before the operation. Post-operative fasting lasted 24 h, during which time two s.c. injections of 6 ml 0.9% saline were given. Long-term post-operative mortality rate was 26% after PBD and 0% after sham operation. Eight months after the operation, all animals were killed by exsanguination under general anesthesia and after fasting for 15 h. For 24 days before being killed, 10 of the 26 PBD animals were randomised to receive the CCK-A receptor antagonist L364,718 (kindly supplied by MSD, West Point, PA) at a dose of 50 µg/kg/h in 70% dimethyl sulfoxide by osmotic mini-pump (Alzet 2002; ALZA, Palo Alto, CA) deposited i.p. The remaining 16 PBD and the 10 sham-operated animals received vehicle by osmotic minipump. The pumps were changed under general anesthesia after 12 days. This resulted in 10 PBD/L364,718-treated animals, 16 PBD animals and 10 shamoperated animals for further study. When the animals were killed, fasting blood samples were collected in EDTA tubes from the inferior vena cava, centrifuged and stored at –70°C until analyzed. Hypercholecystokininemia—nitrosamine study At 10 weeks of age, 43 hamsters received BOP (Ash Stevens, Detroit, MI) s.c. at a single dose of 20 mg/kg body wt. Two weeks after BOP administration, the animals were randomised to undergo either PBD (n 5 28) or sham operation (n 5 15). Long-term post-operative mortality rate was 25% after PBD and 0% after sham operation. Three months (12 weeks) after BOP administration (10 weeks after the operation), all animals were killed by exsanguination under general anesthesia. For 5 days before being killed, eight of the 21 PBD animals were randomised to receive L364,718 as described above. The remaining animals received vehicle. This resulted in eight PBD/ L364,718-treated, 13 PBD and 15 sham-operated hamsters for further study. One hour before being killed, all hamsters received [3H]thymidine through the internal jugular vein (sp. act. 20.0 Ci/mmol; Du Pont Scandinavia AB, Stockholm, Sweden) at a dose of 1 µCi/g body wt.

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Table I. Mean 6 SEM body weight (BW), pancreatic weight (PW), total pancreatic protein and total pancreatic DNA in sham-operated (control), PBD and PBD/L364,718-treated (PBD/L364,718) hamsters 8 months after the operation Group

n BW (g)

PW (mg)

Protein (mg)

DNA (mg)

Control PBD PBD/L364,718

10 116 6 3 16 115 6 2b 10 114 6 4b

343 6 7 446 6 7a 367 6 12b,c

40.4 6 1.6 2.6 6 0.1 52.0 6 1.5a 3.3 6 0.1a b,d 42.6 6 2.1 2.7 6 0.1b,d

, 0.001, bnon-significant compared with control group (Mann–Whitney U-test). c P , 0.001, dP , 0.01 compared with PBD group (Mann–Whitney U-test). aP

Fig. 1. Mean 6 SEM of the fasting plasma CCK and gastrin concentrations in groups of sham-operated (C) (n 5 6), PBD (n 5 6) and PBD/L364,718treated (PBD1A) (n 5 6) hamsters 8 months after the operation. NS, nonsignificant.

Histological analysis and autoradiography The pancreas and all other organs were removed and examined macroscopically. The pancreas was trimmed of adherent fat and weighed and the splenic lobe was fixed in 4% buffered formalin for histological studies. Tissue specimens were embedded in Technovit 7100 plastic (Heraeus Kulzer GmbH, Wehrheim, Germany), cut in 2 µm thick sections and stained with hematoxylin and eosin. Pancreatic lesions, including carcinoma and early putative preneoplastic lesions (tubular ductal complex, cystic ductal complex and intermediate ductal complex), were searched for and classified according to previously described criteria (35–37). The splenic lobe was screened blind for such lesions with a point counting method, using a magnification of 3100 and 100 µm between the points. In each animal, a total of 40 000 points was counted over consecutive visual fields across the tissue sections. For autoradiographical studies in BOP-treated animals, 2 µm thick plasticembedded tissue sections from the splenic lobe were coated with Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), developed after 4 weeks incubation in darkness at 4°C and counterstained with hematoxylin and eosin. The [3H]thymidine labeling index of pancreatic lesions was determined blind at a magnification of 31000. Five or more grains overlying a nucleus was considered significant labeling. In each animal, a total of 3500 labeled and non-labeled nuclei were counted in consecutive areas of the lesions. Labeling index was expressed as a percentage of labeled nuclei. DNA and protein analysis After removing the splenic lobe for histological examination, the rest of the pancreas (gastric and duodenal lobe and head) was weighed, quick frozen and stored at –70°C for DNA and protein analysis. These were analyzed according to the methods described by Labarca and Paigen (38) and Lowry et al. (39) respectively. Plasma gastrin and cholecystokinin assay The concentrations of gastrin in plasma were measured by a specific radioimmunoassay, as previously described (40,41). The concentrations of CCK in plasma were measured by radioimmunoassay using C-terminal-directed antisera without cross-reactivity towards gastrin (42). The assays have been described in detail elsewhere (43,44). Statistical analysis Results are expressed as mean 6 SEM. The two-tailed Student’s t-test as well as the Mann–Whitney U-test were used. Differences were considered significant at P , 0.05 in both tests. When not otherwise mentioned, given values of P are those derived from the Mann–Whitney U-test. Fisher’s exact test was used to evaluate differences in proportions.

Results Long-term hypercholecystokininemia study The fasting plasma concentrations of CCK and gastrin at the time of death are shown in Figure 1. PBD animals had hypercholecystokininemia, whereas plasma gastrin concentrations did not differ significantly between the groups. There were no significant differences in body weight between the groups at the time of death (Table I). The mean pancreatic weight was 30% higher in the PBD group than in the sham316

Table II. Mean 6 SEM body weight (BW), pancreatic weight (PW), total pancreatic protein and total pancreatic DNA in groups of sham-operated (control), PBD and PBD/L364,718 treated (PBD/L364,718) hamsters 12 weeks after a single injection of BOP Group

n

BW (g)

PW (mg)


15 103 6 2 330 6 6 13 101 6 2b 529 6 9a 8 100 6 2b 499 6 11a,c

Protein (mg)

DNA (mg)

37.9 6 1.3 57.3 6 2.1a 53.5 6 2.6a,c

2.4 6 0.1 3.7 6 0.1a 3.2 6 0.1a,c

, 0.001, bnon-significant compared with control group (Mann–Whitney U-test). cNon-significant compared with PBD group (Mann–Whitney U-test). aP

operated group (Table I). The mean total pancreatic content of protein and DNA was increased by 29 and 27% respectively in the PBD group (Table I). No such increases were observed in PBD animals receiving L364,718 over a 24 day period. No focal pancreatic lesions were found upon microscopical examination in PBD and sham-operated hamsters and no macroscopical changes were observed in the liver or other organs. Hypercholecystokininemia—nitrosamine study No significant differences in body weight were seen between the groups at the time of death (Table II). The mean pancreatic weight was 60% higher in the PBD group than in the shamoperated group (Table II). The mean total pancreatic content of protein and DNA was increased by 51 and 54% respectively in the PBD group. PBD animals receiving L364,718 over a 5 day period showed similar increases (Table II). Putative preneoplastic pancreatic lesions, such as tubular ductal complex, cystic ductal complex and intermediate ductal complex, were diagnosed upon microscopical examination in all 21 PBD hamsters, including the animals receiving L364,718. Of the 15 sham-operated animals, four (27%) had such lesions (Table III). Pancreatic ductular carcinoma in situ (Figure 2) was found in two PBD animals. The [3H]thymidine labeling index of pancreatic lesions was significantly higher in the PBD group than in the sham-operated group (Table III and Figure 3). Labeling index did not differ significantly between PBD animals receiving L364,718 and sham-operated animals. No macroscopical changes were observed in the liver or other organs. Discussion PBD did not cause any significant change in the plasma gastrin concentrations, which could have been a source of error. As shown in previous studies (33,34), PBD induces chronic endogenous hypercholecystokininemia with exocrine pancre-

Cholecystokinin and pancreatic carcinogenesis

Fig. 2. Autoradiographed pancreatic section from a BOP-treated, PBD hamster showing ductular carcinoma in situ. 3200.

atic hyperplasia and hypertrophy, which obviously persists after 8 months. This hyperplasia was reversed by infusion of a CCK-A receptor antagonist for 24 days, indicating that the pancreaticotrophic effect of PBD is mediated by CCK. In the study involving BOP, pancreatic growth was not significantly reduced by infusion of the CCK-A receptor antagonist for 5 days, indicating that .5 days are needed to reverse CCKinduced hypertrophy. In a recent study, a trophic effect on the pancreas was observed in hamsters on raw soya diet for 14 days (28). This effect also seemed to be mediated by CCK, since it was blocked by a CCK-A receptor antagonist. However, after 28 days of raw soya feeding, the trophic effect seemed to level off (28). No such tendency was observed after 8 months in the present study and the PBD model thus seems to be preferable for long-term studies. In the rat, PBD over 14 months increased the mean pancreatic weight by 142% (8), which should be compared with 30% in the hamster after 8 months in the present study. Furthermore, long-term PBD in the rat induced premalignant exocrine pancreatic lesions (8,45,46), which were not seen in PBD hamsters after 8 months in the present study. Accordingly, it seems that, as opposed to the rat, the hamster does not develop pancreatic neoplasia on the basis of endogenous hypercholecystokininemia per se. In the hypercholecystokininemia–BOP study, the [3H]thymidine labeling index of the pancreatic lesions was significantly higher in the PBD animals than in the controls, indicating that PBD accelerated progression of the lesions. This was paralleled by a significant increase in the incidence of pancreatic lesions among PBD hamsters. Furthermore, ductular carcinoma in situ was not found in the controls. These findings demonstrate that PBD enhances BOP-induced pancreatic carcinogenesis in the hamster. The increase in the labeling index of the lesions was significantly reduced by infusion of a CCK-A receptor antagonist, suggesting that the promoting effect of PBD is mediated by CCK acting through the CCK-A receptor. The present study is in agreement with observations made by others that CCK and its analogs enhance pancreatic carcinogenesis in the hamster (22–24). However, the issue

is controversial, since some studies with injection of CCK8, CCK-33 or cerulein or feeding with raw soya and trypsin inhibitor showed no effect or even a protective effect on pancreatic carcinogenesis (25–32). Factors should be considered which could explain the difference between those studies and the present one. In some of the previous studies (25–27,29), hypercholecystokininemia was accomplished by injection of CCK-8, CCK-33 or cerulein. PBD causes a persistent and endogenous hypercholecystokininemia, including subfractions of CCK. This may have quite different consequences for pancreatic growth and carcinogenesis than bolus injections of one fraction of CCK or cerulein. However, some studies (28,30–32) using feeding with raw soya or trypsin inhibitor also failed to show an enhancing effect on pancreatic carcinogenesis. In these studies, the body weight of the animals was significantly reduced after long-term raw soya feeding (28,30). Malnutrition may reduce the tendency to develop tumors (47). Furthermore, the present study was designed to investigate the effect of CCK in the early phase of pancreatic carcinogenesis, i.e. during the first 3 months after a single injection of carcinogen. This should be expected to show differences in the incidence of early pancreatic lesions between the experimental and control groups more clearly. Most other studies have investigated the effect of CCK in the late phase of carcinogenesis, which was frequently induced by multiple injections of carcinogen (25–32). In the late stage of carcinogenesis, a high incidence of pancreatic lesions is also seen in the control animals and multiple injections of carcinogen do not allow a clear division into initiation and promotion phases. We conclude that PBD with chronic endogenous hypercholecystokininemia induces pancreatic hyperplasia and enhances BOP-induced early pancreatic carcinogenesis in the hamster. The increases in growth parameters and in [3H]thymidine labeling index of the pancreatic lesions were significantly reduced by administering a CCK-A receptor antagonist, indicating that the trophic and promoting effects of PBD are mediated by CCK acting through the CCK-A receptor. 317

M.Chu, J.F.Rehfeld and K.Borch

Fig. 3. (A) Autoradiographed pancreatic section from a BOP-treated, PBD hamster. There are numerous epithelial cells with nuclear labeling in the ductal complexes. 3200. (B) From a BOP-treated, PBD/L364,718-treated hamster. There are a few labeled epithelial cells in the ductal complexes. 3200.

Table III. Incidence and [3H]thymidine labeling index (LI) of pancreatic lesion in groups of sham-operated (control), PBD and PBD/L364,718-treated (PBD/ L364,718) hamsters 12 weeks after a single injection of BOP Group


n

15 13 8

No. of animals with

LI (%)

Tubular ductal complex

Cystic ductal complex

Intermediate ductal complex

Ductular carcinoma in situ

4 13a 8b

4 13a 8b

4 13a 8b

0 1 1

, 0.001, bP , 0.01 compared with control group (Fisher’s exact test). , 0.01, dnon-significant compared with control group (Mann–Whitney U-test). eP , 0.001 compared with PBD group (Mann–Whitney U-test). aP cP

318

0.6 6 0.2 3.3 6 0.6c 1.2 6 0.2d,e

Cholecystokinin and pancreatic carcinogenesis

Although endogenous hypercholecystokininemia promotes early phase pancreatic carcinogenesis, it does not per se cause development of premalignant or malignant pancreatic lesions in the hamster. Acknowledgements The kind advice of Professor Dr Parviz M.Pour (Department of Pathology, University of Nebraska Medical Center, Omaha, NE) on the histological diagnosis is greatly appreciated. The study was supported by grants from The ¨ stergo¨tland Swedish National Cancer Association and Cancer Funds of O County, Sweden.

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