Cardnogenesis vol.13 no. 11 pp.2047-2052, 1992
Promoting effects of both dietary cholesterol and cholestyramine on pancreatic carcinogenesis initiated by iV-nitrosobis(2-oxopropyl)amine in Syrian golden hamsters
Takahiro Ogawa1, Takao Makino, Keigo Kosahara, Akitoshi Koga and Fumio Nakayama Department of Surgery 1, Kyushu University, Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan 'To whom reprint requests should be sent
Introduction It has become widely accepted that dietary factors, and especially dietary fat intake, are positively associated with the occurrence of cancers of some sites in humans (1 - 3 ) and animals (4—6). Unsaturated fatty acid has also been shown to enhance pancreatic carcinogenesis in rats (7-9) and hamsters (10,11). However, any link between serum levels of cholesterol, another kind of lipid, and cancer incidence remains controversial (12 — 17). Several cohort studies have in fact demonstrated increased incidences of cancer and particularly of colon cancer among men with low serum cholesterol levels (12 — 14). On the other hand, others have shown no such association (15 — 17). Regarding human pancreatic cancer, the data are also equivocal (18-21). The literature on the cancer-cholesterol question also contains inconsistent findings from experimental studies. On the one hand, high cholesterol intake significantly reduced both die time to colon cancer presentation and the survival of dimediylhydrazine treated rats (22) as well as enhancing colon carcinogenesis (23) and remnant gastric carcinogenesis by N-methyl-A^'-nitro-A'nitrosoguanidine in rats (24). On the other hand, a number of •Abbreviations: LCA, lithocholic acid; BOP, /V-nitrosobis(2-oxopropyl)amine; CDCA, chenodeoxycholic acid; HMG-CoA reductase, 3-hydroxy-3-rnethylghrtaryl CoA reductase. © Oxford University Press
Materials and methods Animals Female Syrian golden hamsters (Shizuoka Laboratory Animal Center, Shizuoka, Japan), 6 weeks old and weighing - 8 0 - 1 0 0 g each at the start of the experiment, were used. The animals were housed five per plastic cage in an air-conditioned room at 25°C and 61 % humidity, and were maintained under a daily cycle of 12 h light and dark, and given food and water ad libitum. Qiemicals and diet BOP was provided by Dr Yukio Mori from the Laboratory of Radiochemistry, Gifu Pharmaceutical University, Gifu, Japan. Animals had free access to water and a commercial natural ingredient powder diet containing 0.075% cholesterol (Oriental Yeast Co., Ltd, Tokyo, Japan) or diets containing high doses of cholesterol or cholestyramine. Cholesterol was purchased from Ishizu Chemicals Co., Ltd, Osaka, Japan and Cholestyramine (Quescran*) was from Bristol-Myers Squibb Co., Ltd, Tokyo, Japan. Experimental protocol In a pilot study, a total of 40 hamsters were divided into eight groups. Cholesterol was fed at doses of 0.5 and 1% (w/w). Cholestyramine was fed at doses of 0.5, 1,2,4 and 10% (w/w). After 4 weeks, body and pancreas weights did not show any statistical differences among the groups. The cholesterol contents of the serum, pancreas and liver were significantly increased with both 0.5 and 1 % cholesterol and significantly decreased at cholestyramine doses of 1 % and above. Therefore, doses of 0.5 % for cholesterol and 1 % for cholestyramine were selected for the present carcinogenicity study. A second pilot study was designed to elucidate the subsequent changes in cholesterol content of the serum, pancreas and liver.
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The effects of dietary cholesterol and cholestyramine on pancreatic carcinogenesis initiated with JV-nitrosobis(2oxopropyOamine (BOP) were investigated in 120 female Syrian golden hamsters. BOP (70 mg/kg body weight) was injected s.c. once at the beginning of the experiment. Starting 2 weeks later, the animals were then maintained on basal diet or diets containing either 0.5% cholesterol or 1% cholestyramine for a further 16 weeks. All surviving hamsters were killed at week 18, and the pancreas tissues examined histologically. The incidences of pancreatic carcinomas in hamsters fed cholesterol and the cholestyramine supplement were 40.0 and 30.0% respectively; in both cases significantly higher than the 6.9% incidence in the basal diet group. Cholesterol contents of the serum, pancreas and liver were significantly increased by cholesterol feeding and significantly decreased by the cholestyramine diet. The cholesterol diet also significantly increased pancreatic protein and DNA contents, and the concentration of total bile acids and the level of lithocholk acid in gallbladder bile. The cholestyramine diet significantly increased total pancreatic DNA and protein contents, and pancreatic weight. The results thus indicated that both dietary cholesterol and cholestyramine can enhance BOP-initiated pancreatic carcinogenesis in hamsters.
studies have not confirmed any positive influence on colon (25), breast (26,27), non-operated stomach of rats (24), or skin of mice (28). There is thus the possibility that the relationship between serum cholesterol levels and cancer may depend on the organ or tissue site. No evidence has so far been presented that dietary cholesterol can promote experimental pancreatic carcinogenesis. Cholesterol is an obligatory precursor for bile acids, and dietary cholesterol has been demonstrated to increase the concentration of total bile acids and the level of lithocholic acid (LCA*) in gallbladder bile in hamsters (29,30). Such changes in bile acid metabolism have been shown to enhance pancreatic carcinogenesis in our previous studies (31—33). Lowering of serum cholesterol levels in high-risk subjects and in the population at large has been strongly advocated to reduce coronary heart disease in populations of industrialized countries (34). However, perhaps the greater health concern in view of some epidemiological findings (12—14) is whether lowering blood cholesterol levels will in fact increase die risk of cancer. In order to investigate die effect of lowering serum cholesterol levels on pancreatic carcinogenesis in die present study, hamsters were fed cholestyramine which is a non-absorbable ion-exchange resin with a strong affinity for bile salts. This compound has in fact been shown to promote the induction of colon (35,36) and breast (37,38) cancers in rats, and to increase die development of putative preneoplastic acinar pancreatic lesions in azaserinetreated rats (39). On the odier hand, Cruse et al. (40) found no such effect on colon cancer in rats. The aim of this study was to investigate whether high and low serum cholesterol levels induced by dietary cholesterol and cholestyramine can affect A4utrosobis(2-oxopropyl)arnine (BOP)induced pancreatic carcinogenesis in hamsters.
T.Ogawa et al.
Table I. Experimental details and body, pancreas and liver weight data Group no.
Treatment1
No. of hamsters Initial Effective (final)
Body weight1G 'O Final Initial
1 2 3 4 5 6
Saline - BD Saline - CH Saline - CS BOP - BD BOP - CH BOP - CS
10 10 10 30 30 30
87 ± 89 ± 88* 88 ± 88 ± 89 ±
9(9) 10 (10) 10 (10) 29(29) 30(28) 30(28)
6 6 5 5 6 5
193 205 195 197 205 202
± ± ± ± ± ±
16 16 16 16 18 15
Average daily food intake (g/day)
Pancreas weight per 100 g body weight (g)
Liver weight per 100 g body weight (g)
8.3 8.4 8.1 8.4 8.3 8.5
0.41 0.38 0.44 0.48 0.51 0.50
4.3 6.9 3.0 4.0 7.3 3.5
± .5 ± .0 ± :'.3 ± .8 ± .0 ± .6
± ± ± ± ± ±
0.03 0.03 0.04c 0.06 0.20 0.08
± ± ± ± ± ±
0.4 0.3d 0.3d 0.8 1.5* 0.9=
*BD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. ''Values are expressed as mean ± SD. Significantly different from group 1, P < 0.05. d Significandy different from group 1, P < 0.01. e Signiftcantly different from group 4, P < 0.01. c
Histopathologic observation The splenic, gastric, and duodenal lobes of the pancreas were dissected and fixed in 10% buffered formalin. Liver slices were taken from each lobe. All tissues were processed routinely, embedded in paraffin and sections cut at 4—6 /an were stained with hematoxylin and eosin. Preneoplastic and neoplastic lesions in die pancreas were diagnosed according to the criteria previously described (32,33). Determination of cholesterol contents of the serum, pancreas and liver Samples (50-100 mg) of pancreas and liver were hydrolyzed with 10% KOH in 95% ethanol at 70°C for 2 h. Lipids were extracted with n-hexane. The cholesterol concentration was determined as the trimethylsilyl (TMS) ether derivative by gas-liquid chromatography (GLC) (41,42). Serum cholesterol contents were measured with an autoanaryzer (Olympus-5000, Olympus, Tokyo, Japan) by the method of Liebermann-Burchard (43). Determination of pancreatic protein and DNA Pancreas (200—300 mg) was homogenized in 3 ml ice-cold distilled water using a Potter—Hvehjem homogenizer with a loosely fitting pestle. A 100 /d aliquot was assayed for protein by using die method of Lowry et al. (44) using bovine serum albumin as the standard. A 1 ml aliquot was assayed for DNA by using the method of Schmidt-Thannhauser-Schneider (45) using calf thymus DNA as the standard. Analysis of bile add composition of gallbladder bile In the 4 week feeding pilot study, bile was aspirated from the gallbladder with a 50 /d microsyringe (Hamilton Co., Reno, NE, USA) and 20 pi aliquots were deproteinized with 1 ml of isopropanol. Bile samples corresponding to 2 (J of original bile were hydrolyzed with cholyglycine hydrolase and deconjugated bile acids were analyzed by GLC (Shimazu GC-15A PFsc., Shimazu Co., Kyoto, Japan) as their ethyl ester-dirnethylethylsilyl ether derivatives as reported previously (46). Nordeoxycholic acid (10 jig) was used as an internal standard. For multiple comparisons between the three groups, analysis of variance (ANOVA) was used to determine statistical significance of differences between data expressed as mean ± SD (at P < 0.05) and between incidences of pancreatic lesions by the chi-square test.
Results For the carcinogenicity study, the numbers and body weights of hamsters, and average daily food intakes, as well as pancreas and liver weights at the end of the experiment are shown in Table
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40
100i
400
%
3.0
300
E 2.0
50
1.0-
Serum
25-
Pancreas
Liver
Fig. 1. Effects of feeding diets containing 0.5% cholesterol or 1 % cholestyramine for 4 weeks on cholesterol contents of the serum, pancreas and liver of hamsters. D , Basal diet; E3, 0.5% cholesterol diet; • , 1% cholestyramine diet. Each group consists of five hamsters. Asterisks indicate significant differences (P < 0.01) from the respective basal diet group value.
I. One hamster from group 1 and one from group 4 were not available at final killing due to escape. Two hamsters from group 5 and two from group 6 were killed at week 16 because they became moribund with jaundice due to carcinomas located in the pancreas head. All other animals survived until the end of the experiment. The body weights of the hamsters in groups 1 —6 did not show any statistically significant variation. The pancreas weight in group 3 was significantly increased compared to the group 1 value. No differences in pancreas weight among groups 4—6 were evident. The cholesterol diet significandy increased and cholestyramine significantly decreased liver weight. The data for liver weights demonstrated the same tendencies in the 4 and 8 week pilot studies. Light microsopic examination showed no significant abnormalities in the livers of hamsters fed diets containing cholestyramine. Cholesterol feeding resulted in marked fatty degeneration in the livers. Cholesterol, protein and DNA assay The cholesterol contents of the serum, pancreas and liver were significantly increased by cholesterol feeding and significantly decreased by cholestyramine feeding after 4 weeks (Figure 1). The serum cholesterol levels in each group after 8 and 16 weeks of feeding were not significantly different from the values after 4 weeks. Cholesterol significandy increased pancreatic protein content after 16 weeks of feeding and DNA content after 8 weeks of feeding (Table II). Cholestyramine significantly increased total pancreatic protein content after 16 weeks of feeding and total pancreatic DNA content after 8 weeks of feeding.
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A total of 15 hamsters were divided into three groups fed basal diet or diets containing either 0.5% cholesterol or 1% cholestyramine for 8 weeks. In the caranogenicity study, a total of 120 hamsters were divided into six groups. Groups 1, 2 and 3 served as non-initiated controls receiving a single s.c. injection of 0.9% NaCl followed by basal diet, or diets containing either 0.5% cholesterol or 1 % cholestyramine starting 2 weeks later. Groups 4, 5 and 6 were similarly treated after receiving a single s.c. injection of 70 mg/kg body weight of BOP. Body weights, as well as 1 day food intakes were measured weekly. All but six hamsters were killed at week 18. Animals in all cases were killed under ether anesthesia after fasting for 18 h. Final body, pancreas and liver weights were measured in all experiments. Blood samples as well as pancreas and liver tissues were stored at -20°C until assayed. Serum cholesterol levels were measured in both pilot and carcinogenicity studies. The cholesterol contents of the pancreas and liver, pancreatic protein and DNA values, and the composition of gallbladder bile acids were also measured in the pilot studies.
Promoting effects of cholesterol and cbolestyramine
Table II. Pancreas protein and DNA contents in hamsters fed diets containing 0.5% cholesterol or 1% cholestyramine for 4, 8 and 16 weeks* Treatment1'
Experimental period (weeks)
16
No. of hamsters
Protein content (mg/g pancreas)
Total protein (mg/pancreas)
DNA content (mg/g pancreas)
Total DNA (mg/pancreas)
BD CH CS
5 5 5
136.7 ± 8.2 138.1 ± 13.1 152.7 ± 15.6
60.1 ± 58.8 ± 66.0 ±
5.4 4.7 8.4
3.07 ± 0.22 3.57 ± 0.49 2.88 ± 0.16
1.77 ± 0.01 1.99 ± 0.01 1.63 ± 0.01
BD CH CS
5 5 5
136.3 ± 12.4 153.3 ± 12.5 148.4 ± 11.1
90.4 ± 11.8 98.7 ± 16.0 98.5 ± 17.4
2.49 ± 0.45 4.24 ± 1.02c 3.13 ± 0.51
1.63 ± 0.25 2.36 ± 0.47° 2.05 ± 0.31 c
BD CH CS
9 10 10
132.9 ± 11.1 160.5 ± 21.l c 142.2 ± 10.3
103.0 ± 10.3 116.7 ± 15.4C 124.3 ± 11.6"
1.66 ± 0.49 2.90 ± 0.94 1.75 ± 0.42
1.28 ± 0.42 1.83 ± 0.73 1.54 ± 0.44
Table ffl. Cholesterol concentration and bile acid composition of gallbladder bile in hamsters fed diets containing 0.5% cholesterol or 1% cholestyramine for 4 weeks Treatment'
BD CH CS
Bile acid composition (%)c DCA LCA
CDCA
CA
(ji%l ml)
Concentration of total bile acids Oig/ml)
0.84 ± 0.16 1.58 ± 0.30" 0.69 ± 0.21
1.65 ± 0.14 2.84 ± 0.63" 1.83 ± 0.23
1.4 ± 0.4 2.7 ± 0.6e 1.4 ± 0.3
10.7 ± 3.7 10.9 ± 2.9 4.9 ± 1.0"
68.8 ± 7.6 63.1 ± 7.0 72.4 ± 3.8
No. of hamsters analyzed
Concentration of cholesterol11
5 4 4
19.1 ± 5.8 23.4 ± 4.4 21.4 ± 2.6
*BD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. "Values are expressed as mean ± SD. C LCA, hthocholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid. "Significantly different from the group fed BD, P < 0.05. 'Significantly different from the group fed BD, P < 0.01.
Table IV. Incidences of pancreatic lesions in hamsters fed diets containing 0.5% cholesterol or 1 %cholestyramine Group no.
Treatment*
1 2 3 4 5 6
Saline - BD Saline •- CH Saline •- CS BOP - BD BOP - CH BOP - CS
Effective no. of hamsters
Incidences of pancreatic lesions (%) Gross Adcnocarcinomas tumors
9 10 10 29 30 30
0(0) 0(0) 0(0) 0(0) 7 (23.3)c 6 (20.0)c
0(0) 0(0) 0(0) 2 (6.9) 12 {40.0f 9 (30.0)c
Atypical ductal hyperplasias
Ductal hyperplasias
Adenomas
Total no. of pancreatic lesions per hamster1'
0(0) 0(0) 0(0) 6(20.7) 3 (10.0) 7 (23.3)
0(0) 0(0) 0(0) 9 (31.0) 17 (56.7) 16 (53.3)
0(0) 0(0) 0(0) 0(0) 1 (3.3) 3 (10.0)
0 0 0 0.69 ± 0.85 1.60 ± 1.33" 1.43 ± 0.97"
"BD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. 'Values are expressed as mean ± SD. c Significantly different from group 4, P < 0.05. "Significantly different from group 4, P < 0.01.
Bile acid composition Data for the cholesterol concentration and relative composition of bite acids in gallbladder bile from hamsters fed diets containing cholesterol or cholestyramine are shown in Table HI. Cholesterol significantly increased the concentrations of both cholesterol and total bile acids. Cholesterol significantly increased the value for LCA. Cholestyramine significantly decreased chenodeoxycholic acid (CDCA). Histopathological analysis of pancreatic lesions Histological examination of the pancreas of each group showed the absence of any extracellular fluid, fibrosis, infiltration of
inflammatory cells, and necrosis of acinar cells in the 4 and 8 week pilot studies. In the carcinogenicity study, the incidences and numbers of pancreatic neoplastic and preneoplastic lesions in each group of hamsters are summarized in Table IV. Neither lesions nor inflammatory changes were observed in hamsters from groups 1 - 3 . The incidences of grossly visible tumors in hamsters from groups 5 and 6 were 23.3 and 20.0% respectively, significantly higher than the 0% incidence in hamsters from group 4. The incidences of histopathologically identifiable pancreatic carcinomas in hamsters from groups 5 and 6 were 40.0 and 30.0% respectively, again significantly higher than the 6.9% 2049
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'Values are expressed as mean ± SD. "TJD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. c Significantly different from the BD group, P < 0.05. "Significantly different from the BD group, P < 0.01.
T.Ogawa et al.
incidence apparent in hamsters from group 4. The total numbers of pancreatic lesions per hamster, including adenocarcinomas, atypical ductal hyperplasias, ductal hyperplasias and adenomas, were also significantly higher in groups 5 and 6 than in group 4.
Two main factors could be considered as the cause of pancreatic tumor promotion by cholestyramine. One factor is the observed trophic effect on the pancreas, characterized by increased pancreatic weight, protein and DNA contents as shown in this study. Cholestyramine-induced bile diversion from the intestine is known to raise plasma cholecystokinin levels and promote pancreatic secretion and growth in rats (58,59) and guinea pigs (60). Furthermore, Morgan et al. (39) have indicated that pancreatic trophism exerted by cholestyramine is a possible candidate responsible for the increased development of putative preneoplastic acinar pancreatic lesions in azaserine-treated rats. The other possibility is that lowering cholesterol levels in the serum and pancreas could play a direct role. As cholestyramine is an insoluble resin which is not absorbed through the 2050
Acknowledgements The authors thank Dr Shuichiro Okamoto, Department of Surgery I, Faculty of Medicine, Kyushu University for excellent technical assistance. Thanks are also due to Dr Yukio Mori, Laboratory of Radiochemistry, Gifu Pharmaceutical University, for generously providing BOP.
References 1. Potter.R.D. and McMichael,AJ. (1986) Diet and cancer of the colon and rectum: a case-control study. J. Nail. Cancer Insl., 76, 557-569. 2. Stemmerman.G.N., Nomura.A.M.Y. and Heilbrun.L.K. (1984) Dietary fat and the risk of colorectal cancer. Cancer Res., 44, 4633—4637. 3. Rogers.A.E. and Longnecker.M.P. (1988) Biology of disease. Dietary and nutritional influences on cancer: a review of epidemiologic and experimental data. Lab. Invest., 59, 729-759. 4. Welsch.C.W. (1985) Host factors affecting the growth of carcinogen-induced rat mammary carcinomas: a review and tribute to Charles Brenton Huggins. Cancer Res., 45, 3415-3443. 5. Broitman.S.A., VitaleJ.J., Vavrousek-Jakuba.E. and Gottlieb.L.S. (1977) Polyunsaturated fat, cholesterol and large bowel tumorigenesis. Cancer, 40, 2455-2463. 6. Ip,C. and Sinha.D.K. (1981) Enhancement of mammary tumorigenesis by dietary selenium deficiency in rats with a high polyunsaturatcd fat intake. Cancer Res., 41, 31-34. 7. Longneclcer.D.S., Roebuck,B.D. and Kuhlmann.E.T. (1985) Enhancement of pancreatic carcinogenesis by a dietary unsaturated fat in rats treated with saline or N-nitrosoC2-riydroxyproyI)(2-oxopropyI)amine. / . NatL Cancer hist., 74, 219-222. 8. Woutersen.R A., Garderen-Hoetmer.A., BaxJ. and Scherer.E. (1989)
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Discussion The present results clearly indicated that both cholesterol and cholestyramine promote BOP-initiated pancreatic carcinogenesis in hamsters when supplemented in the diet. To our knowledge this is the first demonstration that both increasing and decreasing serum cholesterol levels might be able to enhance cancer development in the same organ. This result is of great interest in view of the conflicting literature. Dietary cholesterol in the present study significantly increased pancreatic cholesterol, protein and DNA contents. Cholesterol is an important component of cell membranes. Therefore, it has been suggested to be required for the membrane synthesis that occurs earlier than, and perhaps stimulating, DNA replication (47,48). In as much as cell proliferation is a prerequisite for the development of cancer (49,50), there may be a possibility that the increased cholesterol content in the pancreas caused by dietary cholesterol directly enhances pancreatic carcinogenesis by stimulating DNA synthesis. Dietary cholesterol also significantly increased the concentration of total bile acids and the level of LCA in gallbladder bile in this study, in line with earlier reports (29,30). Makino et al. (24) have suggested that an increase in the secretion of bile acids evoked by a high cholesterol diet can enhance carcinogenesis in the remnant stomach, and previous studies suggested that truncal vagotomy acts to increase pancreatic carcinogenesis by elevating total bile acid concentration (33). Furthermore, dietary LCA was found to promote the induction of pancreatic carcinomas in hamsters (32), dietary LCA combined with cholecystectomy exerting the same effect (31). Therefore, changes in bile acid metabolism may be a second factor worthy of attention. In addition, it has been suggested that cholesterol is susceptible to considerable primary oxidation both in vitro and in vivo. For example, cholesterol oxides in blood have been shown to be relatively high in individuals with high serum cholesterol levels (51) and in rabbits fed a high cholesterol diet (52). As cholesterol oxides are known to inhibit the generation of tumor-specific cytotoxic T lymphocytes (53) and to enhance the production of skin cancer in mice (54) and colon cancer in man (55), such a conversion may also be of significance in promoting pancreatic carcinogenesis. Booker et al. (56) have further suggested that dietary cholesterol increases gallbladder prostaglandin synthesis in prairie dogs. As prostaglandin has been indicated as a possible candidate for endogenous promotion of pancreatic carcinogenesis (57), alterations in prostaglandin synthesis could also have played an enhancing role.
gastrointestinal tract, it cannot itself reach target tissues. Therefore, in its promotion of mammary cancer development in rats (37,38), lowering serum lipid levels are implicated. Furthermore, dietary cholestyramine is known to stimulate de novo cholesterogenesis particularly by increasing the activity of 3-hydroxy-3-methylglutaryl (HMG)-fcoA reductase in liver microsomes (61,62). This rate-limiting enzyme of de novo cholesterol synthesis plays an important role in the regulation of cholesterogenesis and is reported to stimulate DNA synthesis and cell proliferation (63,64). Since every nucleated somatic cell regulates de novo cholesterol synthesis (47), this might explain the enhanced pancreatic carcinogenesis observed here. There is one conflicting report in the literature concerning the effects of lowering serum cholesterol levels on experimental pancreatic carcinogenesis. Clofibrate, which is a hypocholesteremic peroxisome proliferator, also causes such reduction while inhibiting A'-nitrosobis(2-hydroxypropyl)amine-initiated pancreatic carcinogenesis in hamsters (65). Clofibrate and its derivatives, however, are known to decrease the activity of HMG-CoA reductase in hamsters and rats (66,67) in direct contrast to the effect of cholestyramine. Reduction in HMG-CoA reductase activity is reported to inhibit DNA synthesis and cell growth in several transformed cell lines (68,69). Therefore, differences in hypocholesteremic mechanisms, in addition to a lack of any trophic effect on the pancreas, could explain the discrepancy wiui cholestyramine. While cholestyramine has a strong affinity for bile salts and eliminates bile salts from the intestine and studies by other investigators have implicated bile acids per se as the cause of colon tumor promotion (35,36), this seems unlikely in the present study, since it exerted little effect on either the concentration of total bile acids or the bile acid composition of gallbladder bile. In conclusion, our results suggest that increasing cholesterol levels in serum and pancreas by dietary cholesterol can enhance pancreatic carcinogenesis. Whether the promotion observed for cholestyramine was simply due to stimulation of pancreatic growth or whether our findings indicate direct modulating effects of lowering cholesterol levels in serum and pancreas are questions presently under investigation in our laboratory.
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Modulation of dietary fat-promoted pancreatic carcinogenesis in rats and hamsters by chronic ethanol ingestion. Carcinogenesis, 10, 453—459. 9. Khoo.D.E., Flaks.B., Oztas.H., Wuliamson,R.C.N. and Habib.N.A. (1991) Effects of dietary fatty acids on the early stages of neoplastic induction in the rat pancreas. Changes in fatty acid composition and development of atypical acinar cell nodules. Int. J. Exp. Path., 72, 571-580. 10. Woutersen.R.A., Garderen-Hoetmer,A., Bax,J., Feringa^A.W. and Scherer.E. (1986) Modulation of putative preneoplastic foci in exocrine pancreas of rats and hamsters. I. Interaction of dietary fat and ethanol. Carcinogenesis, 7, 1587-1593. ll.Birt.D.F., Julius.A.D., White.L.T. and Pour.P.M. (1989) Enhancement of pancreatic carcinogenesis in hamsters fed a high-fat diet ad libitum and at a controlled calorie intake. Cancer Res., 49, 5848-5851. 12. International Collaborative Group (1982) Circulating cholesterol level and risk of death from cancer in men aged 40 to 69 years: experience of an international collaborative group. J. Am. Med Assoc., 248, 2853-2859. 13.Schatzkin,A., Hcover.R.N., Taylor.P.R., Ziegler.R.G., Carter.C.L., Larson.D.B. and Licitra.L.M. (1987) Serum cholesterol and cancer in the NHANES I epidemiologic followup study. Lancet, ii, 298-301. 14. Nomura.A.M.Y., Stemmermann.G.N. and Chyou.P. (1991) Prospective study of serum cholesterol level and large-bowel cancer. J. Nail. Cancer Inst., 83, 1403-1407. 15. Salonen J.T. (1982) Risk of cancer and death in relation to serum cholesterol: a longitudinal study in an Eastern Finnish population with high overall cholesterol level. Am. J. Epidemiol., 116, 622-630. 16. T6mberg,S.A., Holm.L.E., CarstensenJ.M. and Ekhind.G.A. (1986) Risks of cancer of the colon and rectum in relation to serum cholesterol and betalipoprotein. N. Engl. J. Med., 315, 1629-1633. 17. Wald.N.J., Thompson.S.G., Law.M.R., DensenU.W. and Bailey,A. (1989) Serum cholesterol and subsequent risk of cancer results from the BUPA study. Br. J. Cancer, 59, 936-938. 18. Schatzkin.A., Hoover.R.N., Taylor.P.R., Zicgler.R.G., Carter.C.L., Albanes.D., Larson.D.B. and Licitra.L.M. (1988) Site-specific analysis of total serum cholesterol and incident cancer in the National Health and Nutrition Examination Survey I epidemiologic follow-up study. Cancer Res., 48, 452-458. 19.Kromhout,D., Bosschieter.E.B., Drijver.M. and Coulander.C.L. (1988) Serum cholesterol and 25-year incidence of and mortality from myocardial infarction and cancer: The Zutphen study. Arch. Intern. Med., 148, 1051-1055. 20. Howe.G.R., Jain.M. and Miller.A.B. (1990) Dietary factors and risk of pancreatic cancer Results of a Canadian population-based case-control study. Int. J. Cancer, 45, 604-608. 21. Ghadirian.P., Simard.A., BaillargeonJ., Maisormeuve.P. and Boyle.P. (1992) Nutritional factors and pancreatic cancer in the Francophone community in Montreal, Canada. Int. J. Cancer, 47, 1—6. 22. CruseJ.P., Lewin.M.R., Ferulano.G.P. and Clark,C.G. (1978) Cocarcinogenic effects of dietary cholesterol in experimental colon cancer. Nature, 276, 822-825. 23. Klurfeld.D.M., Aglow.E., Tepper.S.A. and Kritchevsky.D. (1983) Modification of dimethylhydrazine-induced carcinogenesis in rats by dietary cholesterol. Nutr. Cancer, 5, 16-25. 24. Makino.M., Kaibara.N. and Koga.S. (1989) Enhanced induction by highcholesterol diet of remnant gastric carcinogenesis by /V-methyl-W-nitro-jVnitrosoguanidine in rats. /. Natl. Cancer Inst., 81, 130-135. 25. Reddy.B.S. and Watanabe.K. (1979) Effect of cholesterol metabolites and promoting effect of lithocholic acid in colon carcinogenesis in germ-free and conventional F344 rats. Cancer Res., 39, 1521-1524. 26. Cohen.L.A. and Chan.P.C. (1982) Dietary cholesterol and experimental mammary cancer development. Nutr. Cancer., 4, 99—106. 27. Klurfeld.D.M. and Kritchevsky.D. (1981) Serum cholesterol and 7,12-dimethylben2[ajanthracene-induced mammary carcinogenesis. Cancer Lett., 14, 273-278. 28. Black.H.S., Henderson.S.V., Kkinhans.C.M., Phelps.A.W. and ThombyJ.I. (1979) Effect of dietary cholesterol on ultraviolet light carcinogenesis. Cancer Res., 39, 5022-5027. 29. Kuroki.S., Muramoto.S., Kurmaoto.T. and HosMta.T. (1983) Effect of feeding cholesterol and sitosterol on hepatic steroid 12or-hydroxylase activity in female hamsters. /. Pharmacobio-Dyn., 6, 551-557. 30. Cohen.B.I., Setoguchi.T., Mosbach.E.H., Mcsherry.C.K., Stenger.R.J., Kuroki.S. and Soloway.R.D. (1987) An animal model of pigment cholelithiasis. Am. J. Surg., 153, 130-138. 31.Ura,H., Makino.T., Ito.S., Tsutsumi.M., Kinugasa.T., Kamano.T., Ichimiya,H. and Konishi.Y. (1986) Combined effects of cholecystectomy and lithocholic acid on pancreatic carcinogenesis of/V-nitrosobis(2-hydroxypropyl) amine in Syrian golden hamsters. Cancer Res., 46, 4782—4786. 32. Makino.T., Obara,T., Ura.H., Kinugasa.T., Kobayashi.H., Takahashi.S. and
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Received on April 15, 1992; revised on July 27, 1992; accepted on August 3, 1992
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58. Brand.S.J. and Morgan,R.G.H. (1982) Stimulation of pancreatic secretion and growth in the rat after feeding cholestyramine. GastroaUerology, 83, 851-859. 59. Koop,!., Undenlhal.M., Schade.M., Trautmann.M., Adler.G. and Arnold^. (1991) Role of cholescystoldnin in cholestyramine-induced changes of the exocrine pancreas. Pancreas, 6, 564—570. 60. Gomez.G., Townsend.C.M., Maani.R., Singh.P., Greeley.G.G. and Thompson J.C. (1989) Down-regulation of pancreatic growth and gallbladder contractility by bile salts. Am. J. Surg. 157, 2 0 - 2 6 . 61. Gonzakz-Pacanowska.D., Marco.C, IglesiasJ., Garcia-MartinezJ. and Garcia-Peregrin.E. (1988) Influence of cholestyramine feeding on mevalonateactivating enzymes. Enzyme, 39, 90—94. 62. Suclding.K.E., Benson.G.M., Bond.B., Gee.A., Glen.A., Haynes.C. and Jackson.B. (1991) Cholesterol lowering and bile acid excretion in the hamster with cholestyramine treatment. Atherosclerosis, 89, 183-190. 63. Siperstein.M.D. (1984) Role of cholesterogenesis and isoprenoid synthesis in DNA replication and cell growth. J. Lipid Res., 25, 1462-1468. 64. Quesney-Huneeus.V., Wiley.M.H. and Siperstein.M.D. (1979) Essential role for mevalonate synthesis in DNA replication. Proc. Natl. Acad. Sri. USA, 76, 5056-5060. 65. Mizumoto.K., Kitazawa.S., Eguchi.T., Nakajima.A., Tsutsumi.M., Ito.S., Denda^A. and Konishi.Y. (1988) Modulation of W-nilrosobis(24rydroxvpropyI) amine-induced carcinogenesis by clofibrate in hamsters. Cardnogenesis, 9, 1421-1425. 66. DeFabian.E., Crestani.M., Malavasi.B., DelPuppo.M., Farina,F., Armocida.C, Bellentani.S., Quack.G. and Bosisio.E. (1989) The effect of etofibrate on cholesterol and bile acid metabolism in the hamster. Pharmacol. Res., 21, 567-576. 67. Bonncfis.D.P.M.T., Rey.C. and Infante.R. (1988) Effects of ciprofibrate and fenofibrate on liver lipids and lipoprotein synthesis in normo- and hyperlipidemic rats. Atherosclerosis, 74, 215—225. 68. Fairbanks.K.P., Barbu.V.D., Wrtte,L.D., Weinstein.I.B. and Goodman.D.S. (1986) Effects of mevinolin and mevalonate on cell growth in several transformed cell lines. J. Cell. Physiol., 127, 216-222. 69. DeClue^.E., Vass.W.C, Papageorge.AJ., Lowy.D.R. and Willumsen.B.M. (1991) Inhibition of cell growth by lovastatin is independent of ras function. Cancer Res., 51, 712-717.
Promoting effects of both dietary cholesterol and cholestyramine on pancreatic carcinogenesis initiated by iV-nitrosobis(2-oxopropyl)amine in Syrian golden hamsters
Takahiro Ogawa1, Takao Makino, Keigo Kosahara, Akitoshi Koga and Fumio Nakayama Department of Surgery 1, Kyushu University, Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan 'To whom reprint requests should be sent
Introduction It has become widely accepted that dietary factors, and especially dietary fat intake, are positively associated with the occurrence of cancers of some sites in humans (1 - 3 ) and animals (4—6). Unsaturated fatty acid has also been shown to enhance pancreatic carcinogenesis in rats (7-9) and hamsters (10,11). However, any link between serum levels of cholesterol, another kind of lipid, and cancer incidence remains controversial (12 — 17). Several cohort studies have in fact demonstrated increased incidences of cancer and particularly of colon cancer among men with low serum cholesterol levels (12 — 14). On the other hand, others have shown no such association (15 — 17). Regarding human pancreatic cancer, the data are also equivocal (18-21). The literature on the cancer-cholesterol question also contains inconsistent findings from experimental studies. On the one hand, high cholesterol intake significantly reduced both die time to colon cancer presentation and the survival of dimediylhydrazine treated rats (22) as well as enhancing colon carcinogenesis (23) and remnant gastric carcinogenesis by N-methyl-A^'-nitro-A'nitrosoguanidine in rats (24). On the other hand, a number of •Abbreviations: LCA, lithocholic acid; BOP, /V-nitrosobis(2-oxopropyl)amine; CDCA, chenodeoxycholic acid; HMG-CoA reductase, 3-hydroxy-3-rnethylghrtaryl CoA reductase. © Oxford University Press
Materials and methods Animals Female Syrian golden hamsters (Shizuoka Laboratory Animal Center, Shizuoka, Japan), 6 weeks old and weighing - 8 0 - 1 0 0 g each at the start of the experiment, were used. The animals were housed five per plastic cage in an air-conditioned room at 25°C and 61 % humidity, and were maintained under a daily cycle of 12 h light and dark, and given food and water ad libitum. Qiemicals and diet BOP was provided by Dr Yukio Mori from the Laboratory of Radiochemistry, Gifu Pharmaceutical University, Gifu, Japan. Animals had free access to water and a commercial natural ingredient powder diet containing 0.075% cholesterol (Oriental Yeast Co., Ltd, Tokyo, Japan) or diets containing high doses of cholesterol or cholestyramine. Cholesterol was purchased from Ishizu Chemicals Co., Ltd, Osaka, Japan and Cholestyramine (Quescran*) was from Bristol-Myers Squibb Co., Ltd, Tokyo, Japan. Experimental protocol In a pilot study, a total of 40 hamsters were divided into eight groups. Cholesterol was fed at doses of 0.5 and 1% (w/w). Cholestyramine was fed at doses of 0.5, 1,2,4 and 10% (w/w). After 4 weeks, body and pancreas weights did not show any statistical differences among the groups. The cholesterol contents of the serum, pancreas and liver were significantly increased with both 0.5 and 1 % cholesterol and significantly decreased at cholestyramine doses of 1 % and above. Therefore, doses of 0.5 % for cholesterol and 1 % for cholestyramine were selected for the present carcinogenicity study. A second pilot study was designed to elucidate the subsequent changes in cholesterol content of the serum, pancreas and liver.
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The effects of dietary cholesterol and cholestyramine on pancreatic carcinogenesis initiated with JV-nitrosobis(2oxopropyOamine (BOP) were investigated in 120 female Syrian golden hamsters. BOP (70 mg/kg body weight) was injected s.c. once at the beginning of the experiment. Starting 2 weeks later, the animals were then maintained on basal diet or diets containing either 0.5% cholesterol or 1% cholestyramine for a further 16 weeks. All surviving hamsters were killed at week 18, and the pancreas tissues examined histologically. The incidences of pancreatic carcinomas in hamsters fed cholesterol and the cholestyramine supplement were 40.0 and 30.0% respectively; in both cases significantly higher than the 6.9% incidence in the basal diet group. Cholesterol contents of the serum, pancreas and liver were significantly increased by cholesterol feeding and significantly decreased by the cholestyramine diet. The cholesterol diet also significantly increased pancreatic protein and DNA contents, and the concentration of total bile acids and the level of lithocholk acid in gallbladder bile. The cholestyramine diet significantly increased total pancreatic DNA and protein contents, and pancreatic weight. The results thus indicated that both dietary cholesterol and cholestyramine can enhance BOP-initiated pancreatic carcinogenesis in hamsters.
studies have not confirmed any positive influence on colon (25), breast (26,27), non-operated stomach of rats (24), or skin of mice (28). There is thus the possibility that the relationship between serum cholesterol levels and cancer may depend on the organ or tissue site. No evidence has so far been presented that dietary cholesterol can promote experimental pancreatic carcinogenesis. Cholesterol is an obligatory precursor for bile acids, and dietary cholesterol has been demonstrated to increase the concentration of total bile acids and the level of lithocholic acid (LCA*) in gallbladder bile in hamsters (29,30). Such changes in bile acid metabolism have been shown to enhance pancreatic carcinogenesis in our previous studies (31—33). Lowering of serum cholesterol levels in high-risk subjects and in the population at large has been strongly advocated to reduce coronary heart disease in populations of industrialized countries (34). However, perhaps the greater health concern in view of some epidemiological findings (12—14) is whether lowering blood cholesterol levels will in fact increase die risk of cancer. In order to investigate die effect of lowering serum cholesterol levels on pancreatic carcinogenesis in die present study, hamsters were fed cholestyramine which is a non-absorbable ion-exchange resin with a strong affinity for bile salts. This compound has in fact been shown to promote the induction of colon (35,36) and breast (37,38) cancers in rats, and to increase die development of putative preneoplastic acinar pancreatic lesions in azaserinetreated rats (39). On the odier hand, Cruse et al. (40) found no such effect on colon cancer in rats. The aim of this study was to investigate whether high and low serum cholesterol levels induced by dietary cholesterol and cholestyramine can affect A4utrosobis(2-oxopropyl)arnine (BOP)induced pancreatic carcinogenesis in hamsters.
T.Ogawa et al.
Table I. Experimental details and body, pancreas and liver weight data Group no.
Treatment1
No. of hamsters Initial Effective (final)
Body weight1G 'O Final Initial
1 2 3 4 5 6
Saline - BD Saline - CH Saline - CS BOP - BD BOP - CH BOP - CS
10 10 10 30 30 30
87 ± 89 ± 88* 88 ± 88 ± 89 ±
9(9) 10 (10) 10 (10) 29(29) 30(28) 30(28)
6 6 5 5 6 5
193 205 195 197 205 202
± ± ± ± ± ±
16 16 16 16 18 15
Average daily food intake (g/day)
Pancreas weight per 100 g body weight (g)
Liver weight per 100 g body weight (g)
8.3 8.4 8.1 8.4 8.3 8.5
0.41 0.38 0.44 0.48 0.51 0.50
4.3 6.9 3.0 4.0 7.3 3.5
± .5 ± .0 ± :'.3 ± .8 ± .0 ± .6
± ± ± ± ± ±
0.03 0.03 0.04c 0.06 0.20 0.08
± ± ± ± ± ±
0.4 0.3d 0.3d 0.8 1.5* 0.9=
*BD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. ''Values are expressed as mean ± SD. Significantly different from group 1, P < 0.05. d Significandy different from group 1, P < 0.01. e Signiftcantly different from group 4, P < 0.01. c
Histopathologic observation The splenic, gastric, and duodenal lobes of the pancreas were dissected and fixed in 10% buffered formalin. Liver slices were taken from each lobe. All tissues were processed routinely, embedded in paraffin and sections cut at 4—6 /an were stained with hematoxylin and eosin. Preneoplastic and neoplastic lesions in die pancreas were diagnosed according to the criteria previously described (32,33). Determination of cholesterol contents of the serum, pancreas and liver Samples (50-100 mg) of pancreas and liver were hydrolyzed with 10% KOH in 95% ethanol at 70°C for 2 h. Lipids were extracted with n-hexane. The cholesterol concentration was determined as the trimethylsilyl (TMS) ether derivative by gas-liquid chromatography (GLC) (41,42). Serum cholesterol contents were measured with an autoanaryzer (Olympus-5000, Olympus, Tokyo, Japan) by the method of Liebermann-Burchard (43). Determination of pancreatic protein and DNA Pancreas (200—300 mg) was homogenized in 3 ml ice-cold distilled water using a Potter—Hvehjem homogenizer with a loosely fitting pestle. A 100 /d aliquot was assayed for protein by using die method of Lowry et al. (44) using bovine serum albumin as the standard. A 1 ml aliquot was assayed for DNA by using the method of Schmidt-Thannhauser-Schneider (45) using calf thymus DNA as the standard. Analysis of bile add composition of gallbladder bile In the 4 week feeding pilot study, bile was aspirated from the gallbladder with a 50 /d microsyringe (Hamilton Co., Reno, NE, USA) and 20 pi aliquots were deproteinized with 1 ml of isopropanol. Bile samples corresponding to 2 (J of original bile were hydrolyzed with cholyglycine hydrolase and deconjugated bile acids were analyzed by GLC (Shimazu GC-15A PFsc., Shimazu Co., Kyoto, Japan) as their ethyl ester-dirnethylethylsilyl ether derivatives as reported previously (46). Nordeoxycholic acid (10 jig) was used as an internal standard. For multiple comparisons between the three groups, analysis of variance (ANOVA) was used to determine statistical significance of differences between data expressed as mean ± SD (at P < 0.05) and between incidences of pancreatic lesions by the chi-square test.
Results For the carcinogenicity study, the numbers and body weights of hamsters, and average daily food intakes, as well as pancreas and liver weights at the end of the experiment are shown in Table
2048
40
100i
400
%
3.0
300
E 2.0
50
1.0-
Serum
25-
Pancreas
Liver
Fig. 1. Effects of feeding diets containing 0.5% cholesterol or 1 % cholestyramine for 4 weeks on cholesterol contents of the serum, pancreas and liver of hamsters. D , Basal diet; E3, 0.5% cholesterol diet; • , 1% cholestyramine diet. Each group consists of five hamsters. Asterisks indicate significant differences (P < 0.01) from the respective basal diet group value.
I. One hamster from group 1 and one from group 4 were not available at final killing due to escape. Two hamsters from group 5 and two from group 6 were killed at week 16 because they became moribund with jaundice due to carcinomas located in the pancreas head. All other animals survived until the end of the experiment. The body weights of the hamsters in groups 1 —6 did not show any statistically significant variation. The pancreas weight in group 3 was significantly increased compared to the group 1 value. No differences in pancreas weight among groups 4—6 were evident. The cholesterol diet significandy increased and cholestyramine significantly decreased liver weight. The data for liver weights demonstrated the same tendencies in the 4 and 8 week pilot studies. Light microsopic examination showed no significant abnormalities in the livers of hamsters fed diets containing cholestyramine. Cholesterol feeding resulted in marked fatty degeneration in the livers. Cholesterol, protein and DNA assay The cholesterol contents of the serum, pancreas and liver were significantly increased by cholesterol feeding and significantly decreased by cholestyramine feeding after 4 weeks (Figure 1). The serum cholesterol levels in each group after 8 and 16 weeks of feeding were not significantly different from the values after 4 weeks. Cholesterol significandy increased pancreatic protein content after 16 weeks of feeding and DNA content after 8 weeks of feeding (Table II). Cholestyramine significantly increased total pancreatic protein content after 16 weeks of feeding and total pancreatic DNA content after 8 weeks of feeding.
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A total of 15 hamsters were divided into three groups fed basal diet or diets containing either 0.5% cholesterol or 1% cholestyramine for 8 weeks. In the caranogenicity study, a total of 120 hamsters were divided into six groups. Groups 1, 2 and 3 served as non-initiated controls receiving a single s.c. injection of 0.9% NaCl followed by basal diet, or diets containing either 0.5% cholesterol or 1 % cholestyramine starting 2 weeks later. Groups 4, 5 and 6 were similarly treated after receiving a single s.c. injection of 70 mg/kg body weight of BOP. Body weights, as well as 1 day food intakes were measured weekly. All but six hamsters were killed at week 18. Animals in all cases were killed under ether anesthesia after fasting for 18 h. Final body, pancreas and liver weights were measured in all experiments. Blood samples as well as pancreas and liver tissues were stored at -20°C until assayed. Serum cholesterol levels were measured in both pilot and carcinogenicity studies. The cholesterol contents of the pancreas and liver, pancreatic protein and DNA values, and the composition of gallbladder bile acids were also measured in the pilot studies.
Promoting effects of cholesterol and cbolestyramine
Table II. Pancreas protein and DNA contents in hamsters fed diets containing 0.5% cholesterol or 1% cholestyramine for 4, 8 and 16 weeks* Treatment1'
Experimental period (weeks)
16
No. of hamsters
Protein content (mg/g pancreas)
Total protein (mg/pancreas)
DNA content (mg/g pancreas)
Total DNA (mg/pancreas)
BD CH CS
5 5 5
136.7 ± 8.2 138.1 ± 13.1 152.7 ± 15.6
60.1 ± 58.8 ± 66.0 ±
5.4 4.7 8.4
3.07 ± 0.22 3.57 ± 0.49 2.88 ± 0.16
1.77 ± 0.01 1.99 ± 0.01 1.63 ± 0.01
BD CH CS
5 5 5
136.3 ± 12.4 153.3 ± 12.5 148.4 ± 11.1
90.4 ± 11.8 98.7 ± 16.0 98.5 ± 17.4
2.49 ± 0.45 4.24 ± 1.02c 3.13 ± 0.51
1.63 ± 0.25 2.36 ± 0.47° 2.05 ± 0.31 c
BD CH CS
9 10 10
132.9 ± 11.1 160.5 ± 21.l c 142.2 ± 10.3
103.0 ± 10.3 116.7 ± 15.4C 124.3 ± 11.6"
1.66 ± 0.49 2.90 ± 0.94 1.75 ± 0.42
1.28 ± 0.42 1.83 ± 0.73 1.54 ± 0.44
Table ffl. Cholesterol concentration and bile acid composition of gallbladder bile in hamsters fed diets containing 0.5% cholesterol or 1% cholestyramine for 4 weeks Treatment'
BD CH CS
Bile acid composition (%)c DCA LCA
CDCA
CA
(ji%l ml)
Concentration of total bile acids Oig/ml)
0.84 ± 0.16 1.58 ± 0.30" 0.69 ± 0.21
1.65 ± 0.14 2.84 ± 0.63" 1.83 ± 0.23
1.4 ± 0.4 2.7 ± 0.6e 1.4 ± 0.3
10.7 ± 3.7 10.9 ± 2.9 4.9 ± 1.0"
68.8 ± 7.6 63.1 ± 7.0 72.4 ± 3.8
No. of hamsters analyzed
Concentration of cholesterol11
5 4 4
19.1 ± 5.8 23.4 ± 4.4 21.4 ± 2.6
*BD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. "Values are expressed as mean ± SD. C LCA, hthocholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid. "Significantly different from the group fed BD, P < 0.05. 'Significantly different from the group fed BD, P < 0.01.
Table IV. Incidences of pancreatic lesions in hamsters fed diets containing 0.5% cholesterol or 1 %cholestyramine Group no.
Treatment*
1 2 3 4 5 6
Saline - BD Saline •- CH Saline •- CS BOP - BD BOP - CH BOP - CS
Effective no. of hamsters
Incidences of pancreatic lesions (%) Gross Adcnocarcinomas tumors
9 10 10 29 30 30
0(0) 0(0) 0(0) 0(0) 7 (23.3)c 6 (20.0)c
0(0) 0(0) 0(0) 2 (6.9) 12 {40.0f 9 (30.0)c
Atypical ductal hyperplasias
Ductal hyperplasias
Adenomas
Total no. of pancreatic lesions per hamster1'
0(0) 0(0) 0(0) 6(20.7) 3 (10.0) 7 (23.3)
0(0) 0(0) 0(0) 9 (31.0) 17 (56.7) 16 (53.3)
0(0) 0(0) 0(0) 0(0) 1 (3.3) 3 (10.0)
0 0 0 0.69 ± 0.85 1.60 ± 1.33" 1.43 ± 0.97"
"BD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. 'Values are expressed as mean ± SD. c Significantly different from group 4, P < 0.05. "Significantly different from group 4, P < 0.01.
Bile acid composition Data for the cholesterol concentration and relative composition of bite acids in gallbladder bile from hamsters fed diets containing cholesterol or cholestyramine are shown in Table HI. Cholesterol significantly increased the concentrations of both cholesterol and total bile acids. Cholesterol significantly increased the value for LCA. Cholestyramine significantly decreased chenodeoxycholic acid (CDCA). Histopathological analysis of pancreatic lesions Histological examination of the pancreas of each group showed the absence of any extracellular fluid, fibrosis, infiltration of
inflammatory cells, and necrosis of acinar cells in the 4 and 8 week pilot studies. In the carcinogenicity study, the incidences and numbers of pancreatic neoplastic and preneoplastic lesions in each group of hamsters are summarized in Table IV. Neither lesions nor inflammatory changes were observed in hamsters from groups 1 - 3 . The incidences of grossly visible tumors in hamsters from groups 5 and 6 were 23.3 and 20.0% respectively, significantly higher than the 0% incidence in hamsters from group 4. The incidences of histopathologically identifiable pancreatic carcinomas in hamsters from groups 5 and 6 were 40.0 and 30.0% respectively, again significantly higher than the 6.9% 2049
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'Values are expressed as mean ± SD. "TJD, Basal diet; CH, diet containing 0.5% cholesterol; CS, diet containing 1% cholestyramine. c Significantly different from the BD group, P < 0.05. "Significantly different from the BD group, P < 0.01.
T.Ogawa et al.
incidence apparent in hamsters from group 4. The total numbers of pancreatic lesions per hamster, including adenocarcinomas, atypical ductal hyperplasias, ductal hyperplasias and adenomas, were also significantly higher in groups 5 and 6 than in group 4.
Two main factors could be considered as the cause of pancreatic tumor promotion by cholestyramine. One factor is the observed trophic effect on the pancreas, characterized by increased pancreatic weight, protein and DNA contents as shown in this study. Cholestyramine-induced bile diversion from the intestine is known to raise plasma cholecystokinin levels and promote pancreatic secretion and growth in rats (58,59) and guinea pigs (60). Furthermore, Morgan et al. (39) have indicated that pancreatic trophism exerted by cholestyramine is a possible candidate responsible for the increased development of putative preneoplastic acinar pancreatic lesions in azaserine-treated rats. The other possibility is that lowering cholesterol levels in the serum and pancreas could play a direct role. As cholestyramine is an insoluble resin which is not absorbed through the 2050
Acknowledgements The authors thank Dr Shuichiro Okamoto, Department of Surgery I, Faculty of Medicine, Kyushu University for excellent technical assistance. Thanks are also due to Dr Yukio Mori, Laboratory of Radiochemistry, Gifu Pharmaceutical University, for generously providing BOP.
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Discussion The present results clearly indicated that both cholesterol and cholestyramine promote BOP-initiated pancreatic carcinogenesis in hamsters when supplemented in the diet. To our knowledge this is the first demonstration that both increasing and decreasing serum cholesterol levels might be able to enhance cancer development in the same organ. This result is of great interest in view of the conflicting literature. Dietary cholesterol in the present study significantly increased pancreatic cholesterol, protein and DNA contents. Cholesterol is an important component of cell membranes. Therefore, it has been suggested to be required for the membrane synthesis that occurs earlier than, and perhaps stimulating, DNA replication (47,48). In as much as cell proliferation is a prerequisite for the development of cancer (49,50), there may be a possibility that the increased cholesterol content in the pancreas caused by dietary cholesterol directly enhances pancreatic carcinogenesis by stimulating DNA synthesis. Dietary cholesterol also significantly increased the concentration of total bile acids and the level of LCA in gallbladder bile in this study, in line with earlier reports (29,30). Makino et al. (24) have suggested that an increase in the secretion of bile acids evoked by a high cholesterol diet can enhance carcinogenesis in the remnant stomach, and previous studies suggested that truncal vagotomy acts to increase pancreatic carcinogenesis by elevating total bile acid concentration (33). Furthermore, dietary LCA was found to promote the induction of pancreatic carcinomas in hamsters (32), dietary LCA combined with cholecystectomy exerting the same effect (31). Therefore, changes in bile acid metabolism may be a second factor worthy of attention. In addition, it has been suggested that cholesterol is susceptible to considerable primary oxidation both in vitro and in vivo. For example, cholesterol oxides in blood have been shown to be relatively high in individuals with high serum cholesterol levels (51) and in rabbits fed a high cholesterol diet (52). As cholesterol oxides are known to inhibit the generation of tumor-specific cytotoxic T lymphocytes (53) and to enhance the production of skin cancer in mice (54) and colon cancer in man (55), such a conversion may also be of significance in promoting pancreatic carcinogenesis. Booker et al. (56) have further suggested that dietary cholesterol increases gallbladder prostaglandin synthesis in prairie dogs. As prostaglandin has been indicated as a possible candidate for endogenous promotion of pancreatic carcinogenesis (57), alterations in prostaglandin synthesis could also have played an enhancing role.
gastrointestinal tract, it cannot itself reach target tissues. Therefore, in its promotion of mammary cancer development in rats (37,38), lowering serum lipid levels are implicated. Furthermore, dietary cholestyramine is known to stimulate de novo cholesterogenesis particularly by increasing the activity of 3-hydroxy-3-methylglutaryl (HMG)-fcoA reductase in liver microsomes (61,62). This rate-limiting enzyme of de novo cholesterol synthesis plays an important role in the regulation of cholesterogenesis and is reported to stimulate DNA synthesis and cell proliferation (63,64). Since every nucleated somatic cell regulates de novo cholesterol synthesis (47), this might explain the enhanced pancreatic carcinogenesis observed here. There is one conflicting report in the literature concerning the effects of lowering serum cholesterol levels on experimental pancreatic carcinogenesis. Clofibrate, which is a hypocholesteremic peroxisome proliferator, also causes such reduction while inhibiting A'-nitrosobis(2-hydroxypropyl)amine-initiated pancreatic carcinogenesis in hamsters (65). Clofibrate and its derivatives, however, are known to decrease the activity of HMG-CoA reductase in hamsters and rats (66,67) in direct contrast to the effect of cholestyramine. Reduction in HMG-CoA reductase activity is reported to inhibit DNA synthesis and cell growth in several transformed cell lines (68,69). Therefore, differences in hypocholesteremic mechanisms, in addition to a lack of any trophic effect on the pancreas, could explain the discrepancy wiui cholestyramine. While cholestyramine has a strong affinity for bile salts and eliminates bile salts from the intestine and studies by other investigators have implicated bile acids per se as the cause of colon tumor promotion (35,36), this seems unlikely in the present study, since it exerted little effect on either the concentration of total bile acids or the bile acid composition of gallbladder bile. In conclusion, our results suggest that increasing cholesterol levels in serum and pancreas by dietary cholesterol can enhance pancreatic carcinogenesis. Whether the promotion observed for cholestyramine was simply due to stimulation of pancreatic growth or whether our findings indicate direct modulating effects of lowering cholesterol levels in serum and pancreas are questions presently under investigation in our laboratory.
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