Early changes in islet hormone secretion in the hamster pancreatic cancer model.

The diabetic state that is seen at a high frequency in association with pancreatic cancer is characterized by elevated plasma levels of several islet hormones and by marked insulin resistance. Both the diabetic state and insulin sensitivity improve after tumor removal by sub-total pancreatectomy. Impaired glucose tolerance has also been found in the hamster pancreatic cancer model, but conflicting data regarding islet function have been reported. In order to further investigate islet function and secretion during early development of pancreatic cancer, we measured the concentrations of insulin, glucagon, somatostatin, and islet amyloid polypeptide (IAPP) in plasma, pancreatic tissue, and secretin-stimulated pancreatic juice at 12 and 27 weeks after the ductal-cell-specific carcinogen, BOP had been used to induce tumors in Syrian golden hamsters. At 12 weeks after BOP, plasma glucagon levels were significantly increased. An exaggerated plasma-glucose response and concomitant hyperinsulinemia were observed at 27 but not 12 weeks after BOP. Plasma IAPP concentrations, but not glucagon or somatostatin, were elevated at 27 weeks. Tissue concentrations of IAPP were substantially reduced in BOP-treated hamsters at 27 weeks. No differences in hormone concentrations were seen in pancreatic juice from the two groups at either of the two time points investigated. The study showed that islet hormone changes accompany the early development of pancreatic tumors in the hamster pancreatic model. The hormone changes and apparent insulin resistance resemble the metabolic changes found in humans with pancreatic cancer.

The intracellular mechanism of insulin resistance in the hamster pancreatic ductal adenocarcinoma model.

Diabetes associated with pancreatic cancer is characterized by profound peripheral insulin resistance. The intracellular mechanism of insulin resistance was investigated in skeletal muscles from N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters. Effects of high-fat diet and exercise also were studied. BOP (20 mg/kg body weight) was administrated weekly for 2 weeks. Hyperinsulinemia was found in BOP-treated hamsters at 20 weeks after BOP treatment, suggesting the peripheral insulin resistance is an early feature in pancreatic cancer. Hamsters were killed at 42 weeks, and soleus muscles were taken for the analysis. Skeletal muscle insulin-receptor binding and insulin receptor tyrosine kinase activities were similar between the control and BOP-treated hamsters. However, maximal muscle glycogen synthase activity was significantly reduced in BOP-treated hamsters compared with the control group. Muscle glycogen phosphorylase activity was increased in the BOP-treated group fed with high-fat diet as well as in BOP-treated groups with exercise. These findings indicate that insulin resistance in the hamster pancreatic cancer model is caused by a postreceptor defect, which led to significant decrease of muscle glycogen synthase activity. Whereas a high-fat diet causes more severe insulin resistance in BOP-treated hamsters, high-fat diet and exercising had no significant effects on skeletal muscle insulin-receptor function and glycogen synthase activity. Furthermore, both high-fat diet and exercise enhanced glycogen phosphorylase activity in BOP-treated hamsters.

Species differences in the expression of transforming growth factor-alpha (TGF-alpha) in the submandibular gland and pancreas.

CONCLUSION: Significant differences exist in the expression of transforming growth factor-alpha (TGF-alpha) in the submandibular glands (SMG) and the pancreas of different species and among cell components in the same species. BACKGROUND: Our previous studies have shown marked differences in the expression of TGF-alpha in the pancreas of humans and Syrian hamsters. To examine whether these differences also exist in other species, we examined the expression of TGF-alpha in the pancreas of mouse, rat, Syrian hamster, guinea pig, rabbit, pig, dog, and monkey. We included the SMG of these species for comparison. MATERIALS AND METHODS: The formalin-fixed tissues of these species (n = 3) were investigated by immuno-histochemistry using a monoclonal antibody to TGF-alpha. The SMG of rat, mouse, hamster, rabbit, pig, dog, and monkey were examined by RT-PCR to assure the specificity of the antibody. RESULTS: Remarkable species differences were found in the expression of this peptide in both the SMG and the pancreas. In the SMG, the expression varied in different cell components, even in the same tissue of the species. Although excretory and secretory ducts of the SMG of most species reacted with the antibody, intercalated ducts were immunoreactive only in mouse and guinea pig. Acinar cells were either weakly positive or nonimmunoreactive. In the pancreas of most species, the cells of the large and medium-sized ducts expressed TGF-alpha, whereas centroacinar cells of only rat and dog reacted with the antibody. Marked differences were found in the expression of TGF-alpha in islet cells and in its spatial distribution. Differences were also found in the immunoreactivity of mesenchymal and neural cells among the species.

The effect of streptozotocin and a high-fat diet on BOP-induced tumors in the pancreas and in the submandibular gland of hamsters bearing transplants of homologous islets.

The effect of a high-fat diet (HF) and streptozotocin (STZ) was investigated in the rapid cancer induction model developed in our laboratories. Syrian golden hamsters bearing homologous islets transplanted in their right submandibular gland (SMG) received a HF or a low-fat diet (LF). Half of the animals from each dietary group received STZ (HF-STZ and LF-STZ groups) and the other half did not (HF and LF groups). One week later, all hamsters were treated with N-nitrosobis(2-oxopropyl)amine (BOP) weekly for 3 weeks and the experiment was terminated 12 weeks after the last BOP injection. Pancreatic lesions were found in many hamsters, with a lower incidence in the LF-STZ group (13%) than in other groups (35-45%). A HF diet counteracted the inhibitory effect of STZ on pancreatic tumor induction by yet unknown mechanisms. SMG tumors, all ductal-type adenocarcinomas, developed in all groups and the incidence was lowest in the HF group (6%) compared to the LF group (15%), LF-STZ group (17%) and HF-STZ group (18%). However, the difference was not statistically significant. It was concluded that a HF diet counteracts the inhibitory effect of STZ on BOP-induced pancreatic lesions but has no effect on the induction of tumors in the SMG. STZ pretreatment does not influence tumor induction of the SMG of these hamster.

Experimental evidence for the origin of ductal-type adenocarcinoma from the islets of Langerhans.

To investigate the role of the islets of Langerhans in pancreatic carcinogenesis, freshly isolated islets from male Syrian hamsters were transplanted into the right submandibular glands of 50 female hamsters that were or were not pre-treated with streptozotocin. Thyroid gland fragments, cellulose powder, and immortal hamster pancreatic ductal cells were injected into the left submandibular gland of the same hamsters. All recipient hamsters were then treated with the potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine weekly at a dose of 40 mg/kg of body weight for 3 weeks. Between 3 and 8 weeks later, 18 of 75 (24%) hamsters developed large ductal-type adenocarcinomas in the submandibular gland region, where islets were transplanted, but none developed tumors in the left submandibular gland. In 9 of 18 hamsters, tumors were multiple so that a total of 31 cancers were found. Eleven of these carcinomas were in the vicinity of transplanted islets, eight of which showed intra-insular ductular or cyst formation as seen in the pancreas of hamsters during pancreatic carcinogenesis. The formation of ductular structures within islets was also demonstrated in vitro. Some tumor cells in the vicinity of these islets were reactive with anti-insulin. Y chromosome message was found by polymerase chain reaction analysis in one of the three tumors examined. Also, like the induced pancreatic tumors, all three submandibular gland tumors that were examined had the mutation of the c-Ki-ras oncogene at codon 12 and all tumors expressed blood group A antigen. These and other findings strongly suggest that some components of islets, most probably stem cells, are the origin of ductal-type adenocarcinomas in this model.

Effects of high-fat diet and cholecystokinin receptor blockade on promotion of pancreatic ductal cell tumors in the hamster.

The mechanism by which high-fat diets potentiate pancreatic cancer is not known, but pancreaticotrophic hormones such as cholecystokinin (CCK) may be involved. The effect of CCK receptor blockade on carcinogenesis during the entire promotion period was investigated in Syrian Golden hamsters fed a high- or low-fat diet and treated with N-nitrosobis(2-oxopropyl)amine (3 x 10 mg/kg at weekly intervals). One-half of the hamsters fed a high-fat diet received the CCK-A receptor antagonist devazepide (25 nmol/kg/hr) for the duration of the experiment. At 39 weeks the incidence of pancreatic malignancies was significantly higher in hamsters fed the high-fat diet than in those fed the low-fat diet (p < 0.05). Tumor incidence was not changed by CCK receptor blockade. Potentiation of pancreatic cancer by a high-fat diet in hamsters does not appear to be influenced by endogenous CCK during the tumor promotion period.

Stimulation of islet cell proliferation enhances pancreatic ductal carcinogenesis in the hamster model.

Previous studies have shown that some N-nitrosobis (2-oxopropyl)amine (BOP)-induced ductal/ductular pancreatic cancers in the hamster model develop within islets and that streptozotocin (SZ) pretreatment that caused islet degeneration and atrophy inhibits pancreatic cancer induction. Hence, it appears that in this model islets play a significant role in exocrine pancreatic carcinogenesis. To examine whether stimulation of islet cell proliferation (nesidioblastosis) enhances pancreatic exocrine cancer development, we tested the effect of the pancreatic carcinogen BOP in hamsters after induction of nesidioblastosis by cellophane wrapping. Before wrapping, hamsters were treated with SZ to inhibit pancreatic tumor induction in the unwrapped pancreatic tissues. Control groups with a wrapped pancreas did not receive SZ. Six weeks after SZ treatment, all hamsters were treated with BOP (10 mg/kg body weight) weekly for 10 weeks and the experiment was terminated 38 weeks after the last BOP treatment. Many animals recovered from their diabetes at the time when BOP was injected and many more after BOP treatment. Only nine hamsters remained diabetic until the end of the experiment. Both SZ-treated and control groups developed proliferative and malignant pancreatic ductal-type lesions primarily in the wrapped area (47%) but less frequently in the larger segments of the pancreas, including the splenic lobe (34%), gastric lobe (13%), and duodenal lobe (6%). Only a few lesions developed in the unwrapped pancreatic region of nine diabetic hamsters with atrophic islets, whereas seven of these hamsters had tumors in the wrapped area. Histologically, most tumors appeared to originate from islets, many invasive carcinomas had foci of islets, and some tumor cells showed reactivity with anti-insulin. The results show that, in the BOP hamster model, islets are the site of formation of the major fraction of exocrine pancreatic cancer and that induction of nesidioblastosis enhances pancreatic carcinogenesis.

Effects of voluntary physical exercise on high-fat diet-promoted pancreatic carcinogenesis in the hamster model.

We examined the effect of voluntary physical exercise (running wheels) on pancreatic carcinogenicity of N-nitrosobis-(2-oxopropyl) amine (BOP) in groups of female Syrian hamsters fed a high-fat (HF) diet in which corn oil was 24.6% of the diet or a low-fat (LF) diet in which corn oil was 4.5% of the diet. Each group was divided into an exercising (EX) group (LF-EX and HF-EX) and a sedentary (S) group (LF-S and HF-S). All hamsters were treated with BOP (20 mg/kg body wt) weekly for two weeks beginning four weeks after the experimental diets, which were fed from weaning. A modified glucose tolerance test was performed before the BOP injections and then again at 20 and 40 weeks, and the levels of glucose, insulin-like growth factor I, and insulin were determined in the plasma samples. At the end of the experiment, serum levels of lipid metabolites were also examined in six hamsters from each group. The experiment was terminated 44 weeks after the BOP treatment. Pancreatic ductal/ductular adenocarcinoma incidence was significantly higher in hamsters fed the HF diet (HF-S and HF-EX) than in those fed the LF diet (LF-S and LF-EX). In all groups, glucose and insulin-like growth factor I levels remained within the normal range throughout the experiment, whereas insulin and lipid metabolite levels were significantly elevated in all hamsters fed the HF diet (HF-S and HF-EX). Exercise significantly reduced the insulin level in the group fed the HF diet but did not influence the cancer burden, possibly by the generation of reactive lipid metabolites. Overall, the results showed that voluntary physical exercise does not influence the promotional action of the HF diet on pancreatic carcinogenesis in hamsters. This action could be attributed to the ability of the HF diet to increase the secretion of insulin, which has a growth-promoting and mitogenic effect on pancreatic cells, and to the effect of an HF diet or physical exercise in producing excess free radicals.

Cellular and subcellular localization of transforming growth factor-alpha and epidermal growth factor receptor in normal and diseased human and hamster pancreas.

Four normal pancreas, 8 chronic pancreatitis specimens, and 30 non-endocrine pancreatic tumors from humans and 6 normal and 6 induced pancreatic cancers in hamsters were examined immunohistochemically by antibodies against human transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR). Two normal pancreas and two pancreatic cancer specimens from each species were also studied immunoelectron microscopically by the immunogold method. In chronic pancreatitis, the reactivity and intensity of the staining with both antibodies were much greater in ductal/ductular cells than in the normal pancreas. All 30 pancreatic cancers reacted with both antibodies with a variable degree of reactivity and staining intensity. No correlation was found between the histological type of tumors, the degree of tumor differentiation, and the incidence and patterns of reactivity of either antibody. Immunoelectron microscopically, both EGFR and TGF-alpha were demonstrated primarily on the basal membrane. In the normal hamster pancreas, TGF-alpha was overexpressed in the alpha-cells but not in any other islet cells. Both TGF-alpha and EGFR were marginally detectable in the exocrine pancreas and in induced pancreatic lesions. This is the first demonstration of subcellular localization of TGF-alpha and EGFR in the normal and diseased human and hamster pancreas.

A new technique for simultaneous demonstration of 4 tumor-associated antigens in pancreatic cancer cells.

A new technique is presented for demonstration of 4 tumor-associated antigens in a single slide prepared from paraffin-embedded specimens that can be kept permanently. By sequential processing of the slides with monoclonal antibodies B72.3, DU-PAN-2, CO19-9 and CO17-1A, immunoreactivity with all 4 antibodies can be visualized. Examination of parallel slides stained only with one of the antibodies revealed no interference of the reactive products of the 4 antibodies with each other. Some tumor cells demonstrated reactivity with two antibodies. The described technique provides a possibility to investigate spatial distribution of antigens in tumor cells, and it may be useful for diagnosis, prediction of prognosis and a more appropriate tumor classification.

Effects of raw soya diet and cholecystokinin receptor blockade on pancreatic growth and tumor initiation in the hamster.

Raw soya diet in the hamster had short-term trophic effects on the pancreas, causing significant increases in pancreatic weight, DNA, RNA, and protein. These changes appear to be mediated by cholecystokinin (CCK) because the increases were blocked by infusion of the CCKA receptor antagonist, MK329. Raw soya diet significantly increased plasma levels of CCK in both the short-term and long-term studies. However, raw soya did not potentiate pancreatic cancer in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Infusion of MK329 during the initiation period of carcinogenesis did not change tumor incidence or yield, suggesting that endogenous CCK does not influence tumor induction during the initiation period in the hamster.

Involvement of cytochrome P450 2E1-like isoform in the activation of N-nitrosobis(2-oxopropyl)amine in the rat nasal mucosa.

Induction of tumours in the nasal olfactory region of MRC rats by N-nitrosobis(2-oxopropyl)amine (BOP) is inhibited by orchiectomy and restored by testosterone. These results suggest the involvement of a sex-specific enzyme in BOP bioactivation in rat nasal mucosa. The present study was undertaken to identify this enzyme. Enzyme-linked immunosorbent assay (ELISA) and the metabolism of known substrates (p-nitrophenol) pointed to a microsomal cytochrome P450 (P450) 2E1-like isoform as a candidate enzyme. A correlation was found between the enzyme activity in nasal mucosal microsomes and serum testosterone levels. Four times more activity was detected in the nasal mucosa than in the liver of male rats. Vanillin inhibited the activity of the nasal mucosal enzyme to a greater extent than that of the liver enzyme. The overall results suggest that a nasal mucosal P450 2E1-like isoform is involved in BOP metabolism.

The patterns of coexpression of tumor-associated antigens CA 19-9, TAG-72, and DU-PAN-2 in human pancreatic cancer.

Coexpression of CA 19-9, DU-PAN-2, and TAG-72 was examined by a multilabeling immunohistochemical procedure in 31 surgically resected human pancreatic carcinomas. CA 19-9 was expressed in 74%, DU-PAN-2 in 84%, and TAG-72 in 65% of the cases. CA 19-9 and DU-PAN-2 were coexpressed in 16 cases (52%), CA 19-9 and TAG-72 in 10 cases (32%), DU-PAN-2 and TAG-72 in 8 cases (26%), and all three antigens in 10 tumors (32%). With the combination of the three antibodies, all 31 tumors were labeled. However, heterogeneity in antigen expression existed and the antibodies against CA 19-9, DU-PAN-2, and TAG-72 depicted 55%, 49%, and 35% of the tumor cells, respectively. The average number of cells coexpressing CA 19-9 and DU-PAN-2, CA 19-9 and TAG-72, DU-PAN-2, and TAG-72 was 22%, 11%, and 10%, respectively. Only about 3% of tumor cells coexpressed all three antigens, whereas 8% of tumor cells did not express any of the antigens. There was no correlation between the patterns of antigen expression and age or sex. However, there was a tendency of reduced CA 19-9, DU-PAN-2, and TAG-72 expression in less differentiated tumor areas. The results show that: 1) pancreatic cancer cells coexpress two or three antigens in different proportions; and 2) although the sensitivity for pancreatic cancer reaches 100% by all three antibodies, a remarkable heterogeneity exists and a minor fraction of tumor cells does not seem to produce any of these antigens.

Endocrine aspects of exocrine cancer of the pancreas. Their patterns and suggested biologic significance.

Nine human exocrine pancreatic adenocarcinomas were examined by serial sectioning and double- and triple-labeled immunohistochemical techniques with antibodies against chromogranin A, insulin, islet amyloid polypeptide, glucagon, somatostatin, pancreatic polypeptide, serotonin, pancreastatin, and neuron-specific enolase. The results were correlated with the stage of the disease, histologic characteristics of the tumors, and survival of the patients. Cells immunoreactive with most or all of the antibodies were found in all nine cases. Abnormal co-location of some hormones in the same cell and the lack of normal co-location of other hormones were found. Endocrine cells also were identified in the invasive regions of the cancer, including perineural spaces. Abnormality in the production and release of the peptide was indicated not only in the endocrine cells of exocrine cancer, but also in the islets near the cancer. Patients whose cancer contained many endocrine cells seemed to survive longer than those with tumors containing fewer endocrine cells. The overall data suggested that the observed abnormalities may contribute to the impaired glucose tolerance found in six of these patients.

Effects of high fat diet and cholecystokinin receptor blockade on pancreatic growth and tumor initiation in the hamster.

The mechanism by which high-fat diet potentiates pancreatic cancer is not known, but trophic hormones may be involved. In preliminary growth studies, hamsters fed a high fat diet (17.5% lard, 17.5% corn oil) for 14 days showed a 16.3% increase (P < 0.01) in pancreatic weight compared to controls on low fat diet (2.5% lard, 2.5% corn oil). A significant increase was also seen at 28 days. Similar increases were seen in pancreatic DNA (29%, P < 0.01) and pancreatic RNA (22%, P < 0.05) at 14 days. Plasma cholecystokinin (CCK) levels at 14 days were 2.5 fold higher in the animals fed high fat (P < 0.01). Infusion of the CCK antagonist MK329 (25 nmol/kg/h) completely abolished the increase in pancreatic weight, pancreatic DNA and pancreatic RNA. The effect of CCK receptor blockade during the initiation period of carcinogenesis was investigated in hamsters fed the same diets used in the growth studies. One hundred animals received a single injection of N-nitrosobis(2-oxopropyl)amine, (BOP, 20 mg/kg). Half of the hamsters in each diet group received a 2 week infusion of MK329 (25 nmol/kg/h), beginning 8 days before carcinogen administration. At the time of death, 55 weeks after carcinogen administration, non-fasting plasma CCK levels were 31% higher in the high fat fed hamsters than in the low fat fed animals (P < 0.01). The high-fat diet group had a 3-fold increase in total cancer incidence and a 5-fold increase in advanced lesions (adenocarcinomas). Tumor incidence and yield were not changed in either diet group by CCK-receptor blockade during the initiation period. Cholecystokinin appears to mediate the short-term trophic effect that high-fat feeding has on the pancreas. However, potentiation of pancreatic cancer by high-fat diet in the hamster cancer model does not appear to be influenced by endogenous cholecystokinin at the time of tumor induction.

A new multilabeling technique for simultaneous demonstration of different islet cells in permanent slides.

We describe a simple immunohistochemical method for simultaneous demonstration of insulin, glucagon, somatostatin, and pancreatic polypeptide cells of the Langerhans islet in routinely fixed paraffin-embedded human and Syrian hamster pancreases. With the use of alcohol insoluble chromogens, the slides can be mounted in Permount and be kept permanently without losing the color quality and intensity. With this technique, the spatial topographical distribution and morphology of the individual pancreatic endocrine cells can be investigated in the healthy and diseased pancreas.

Modification of antigen expression in human and hamster pancreatic cancer cell lines induced by sodium butyrate.

The effects of sodium butyrate (NaB) on the growth, morphology, and expression of blood group A, Lewis(a), and CA 19-9 antigen in the hamster pancreatic cancer cell lines, PC-1 (well differentiated) and PC-1.0 (poorly differentiated), and of blood group A, DU-PAN-2, and CA 19-9 antigens in four human pancreatic cancer cell lines, HPAF and CD11 (well differentiated) and CD18 and PANC-1 (poorly differentiated), were examined. NaB inhibited the growth of all cell lines and induced cell enlargement, an increase in secretory material, microfilaments, and pseudopodia. NaB stimulated the production of blood group A antigen in PC-1.0 cells dose dependently, but no change in the expression of this antigen was observed in the human cell lines. However, NaB treatment increased the presence of cells positive for CA 19-9 in PANC-1 but not in the remaining cell lines, none of which reacted with the anti-CA 19-9 antibody before or after NaB treatment. Untreated PANC-1 cells did not produce either blood group A or DU-PAN-2 antigen, but expressed these antigens after NaB treatment in a dose-dependent manner. The results suggest that NaB stimulates the differentiation of the hamster and human pancreatic cancer cell lines and increases or induces the expression of some tumor-associated antigens.

Relationship between blood group-A antigen expression and malignant potential in hamster pancreatic cancers.

The loss of expression of the ABH blood group antigens is suggested to be associated with more aggressive behavior of cancers. We have compared the growth behaviors of two hamster pancreatic cancer cell lines with different blood group-A expressions. PC-1.0 cells, which expressed blood group-A antigen poorly, showed a faster growth in vitro and in vivo when implanted into the pancreas of homologous animals, whereas PC-1.2 cells, all of which express the antigen, had a slower growth rate both in vitro and in vivo. PC-1.0 also tended to metastasize, whereas PC-1.2 cells grew primarily locally. The allografts of both PC-1.2 cells (PC-1.2AG) and PC-1.0 cells (PC-1.0AG) and the metastases of PC-1.0 cells expressed blood group A antigen in a similar rate. There was no significant difference in the number of A-antigen positive cells (A+) between the PC-1.2AG and PC-1.0AG, although the expression of A antigen in PC-1.0AG showed a greater heterogeneity. The combined immunohistochemistry and autoradiography did not show any significant differences in the labeling index of A+ or A- cells between the two allografts. Thus, the results indicate that blood group A antigen expression is unrelated to malignancy in this model. The faster growth rate of PC-1.0 cells may be due to their shorter cell cycle.

Submandibular gland as a site for islet transplantation.

Homologous transplantation of islets of Langerhans into the submandibular glands of Syrian hamsters was successful in 8 out of 10 recipients. The technique was simple and led to formation of islets of various sizes within the parenchyma of the gland. The morphology and endocrine cell patterns of this islets were identical to pancreatic islets. The advantage of this model for islet transplantation is discussed.

Cholecystokinin inhibits DNA alkylation induced by N-nitrosobis (2-oxopropyl)amine (BOP) in hamster pancreas.

Cholecystokinin (CCK) inhibits pancreatic cancer but not hepatic tumor induction by N-nitrosobis (2-oxopropyl) amine (BOP) in hamsters when administered with or shortly before BOP. In this study, we evaluated the capability of sulfated CCK-8 to inhibit DNA alkylation in the hamster pancreas. We examined the pattern of O6-methylguanine (G6-Me) and N7-methylguanine (G7-Me) in pancreatic ductal, acinar and liver tissues from Syrian hamsters treated with a single dose of BOP (20 mg/kg s.c.) and with five s.c. injections of CCK-8 (200 pM/kg, 30 min apart). The first CCK injection was given either 90 min before, or together, or 3 h after POP administration. The amount of G6-Me in liver DNA did not differ significantly. We observed a decrease of G7-Me in the liver of the group treated with CCK together with POP as compared to POP alone (P less than 0.005). Lower amounts of G6-Me were found in ductal preparations (P less than 0.01) of the animals treated with CCK before POP as compared to POP alone. CCK also modified the pattern of alkylation in the acinar tissue, but without a clear relationship with the timing of administration. The results suggest that the inhibitory effect of CCK-8 on pancreatic carcinogenicity of BOP could be related to its capability to modify DNA alkylation by yet unknown mechanisms.