Reduction of colonic carcinogenesis by wheat bran independent of fecal bile acid concentration.

It has been reported previously that populations with a decreased concentration of fecal bile acids have a lower incidence of colon cancer. We examined the importance of fecal bile acid dilution by wheat bran (WB) in inhibiting colonic tumorigenesis in an experimental animal model. Male F344 rats received oral doses of the colon carcinogen 1,2-dimethylhydrazine [CAS: 540-73-8] and were assigned randomly to groups fed one of four semipurified diets for 26 weeks. The diets were fiber-free (FF), 10% WB, FF + bile salts, or WB + bile salts. The amount of bile salts added was adjusted to produce a fecal bile acid concentration in the group fed WB + bile salts equal to that found in the FF groups. Fecal bile acid concentrations at 12 and 24 weeks in the WB + bile salts group were similar to those in the FF group. Gross and microscopic findings at necropsy revealed a reduced total number and multiplicity of colon tumors in both bran-fed groups. Although the fecal bile acid concentrations of the FF and WB + bile salts groups were equal, the latter showed a significant reduction in tumor yield.

Dietary fiber-mediated mechanisms in carcinogenesis.

Dietary fiber may affect the development of cancers of the gastrointestinal tract and the breast. The biological intermediates studied most have been fecal bile acids; both human and animal studies suggest a tumor-promoting role of bile acids in the development of colon tumors, although there are conflicting data from human studies. Short-chain fatty acids are major fermentation products of bacterial degradation of dietary fiber. If short-chain fatty acids explained the tumor-inhibiting properties of dietary fiber, the readily fermentable fibers such as guar and pectin would be more protective than cellulose and wheat bran, which has not been observed. Because these two hypotheses do not adequately explain modulation of tumor growth by dietary fiber, investigation of other intermediates is indicated. These include physical characteristics of the feces, such as abrasiveness; intestinal microflora; aqueous-phase bile acids, which may represent the bioavailable pool; alterations in mucins; mutagenicity of intestinal contents; alterations in mucosal cytokinetics; activities of enzymes, such as ornithine decarboxylase or aryl hydrocarbon hydroxylase; neurogenic effects caused by changes in intestinal bulk or short-chain fatty acids; gut hormones or other peptide growth factors (local or systemic); enterohepatic circulation of hormones; transit time; pH; or decreased availability of total dietary energy.

Dietary fat versus caloric content in initiation and promotion of 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats.

Enhancement of mammary tumor formation by dietary fat may be mediated via increased caloric intake. Three experiments were performed to study this relationship in 7,12-dimethyl-benz(a)anthracene (DMBA)-treated female Sprague-Dawley rats: (a) high- or low-fat isocaloric diets were fed in a crossover design; (b) low-fat, high-calorie and high-fat, low-calorie diets were fed in a crossover design; (c) pair-fed rats were restricted to 60% of the calories of controls with ad libitum access to food beginning 10 days after DMBA administration. The pair-fed rats received daily 60% of calories, the same level of fiber, and 115% more fat than did rats fed ad libitum. Tumor yield but not tumor incidence was greater in rats fed high-fat rather than low-fat isocaloric diets prior to initiation of tumorigenesis. A low-fat, high-calorie diet led to more tumor incidence and yield than was associated with feeding of a high-fat, low-calorie diet. Caloric restriction (although with concomitant intake of more fat) led to complete inhibition of tumor formation. These results indicate that both high-fat and high-calorie diets exhibit cocarginogenic, not merely promotional, properties. Caloric intake may be a greater determinant than dietary fat of a tumor-enhancing regimen. Finally, restriction of caloric intake during promotion markedly suppresses tumor formation, despite the increased fat content of the restricted diet, suggesting a permissive role for calories in tumor formation. The possibility remains that alterations in levels of other dietary components could also have contributed to the observed effects.

Inhibition of chemically induced mammary and colon tumor promotion by caloric restriction in rats fed increased dietary fat.

Tumor promotion associated with increased dietary fat may be inhibited by reduction in total caloric intake. This hypothesis was tested in rats given either 7,12-dimethylbenz(a)anthracene to induce mammary tumors or 1,2-dimethylhydrazine to induce colon tumors. One week after dosage with either carcinogen, the rats were fed semipurified diets that provided 4% fat with ad libitum calories or 13.1% fat with a reduction of calories by 40% from ad libitum intake. Rats treated with 7,12-dimethylbenz(a)anthracene and subjected to caloric restriction weighed 40% less than those fed ad libitum; rats treated with 1,2-dimethylhydrazine were heavier at the onset of caloric restriction and lost weight and weighed approximately 40% less than animals fed ad libitum. At 20 weeks after 7,12-dimethylbenz(a)anthracene administration, rats fed ad libitum had 80% tumor incidence while in those fed restricted calories, 20% had tumors (P less than 0.001). All other measures of mammary tumor growth were significantly reduced in rats given restricted calories. Six months after 1,2-dimethylhydrazine administration, colon tumor incidence was 100% in rats fed ad libitum and 53% in those fed the calorie-restricted diet (P less than 0.001). This reduction of colonic carcinogenesis was seen despite a significant increase in mucosal labeling index following [3H]thymidine autoradiography. This paradoxical finding may be due to the increased fat content of the calorie-restricted diet. These data demonstrate that the tumor-promoting effects of dietary fat can be more than offset by a reduction in total caloric intake and that the promoting effect of fat may be due, at least in part, to its greater caloric density.

Caloric restriction and 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats: alterations in circulating insulin, insulin-like growth factors I and II, and epidermal growth factor.

Caloric restriction (CR) inhibits many neoplastic diseases in rodents, yet the biochemical mechanism(s) for these effects are poorly understood. We have examined the effects of ad libitum (AL) feeding with 25 or 40% CR on the promotion of 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in virgin female Sprague-Dawley rats. Further, we have also studied the influence of chronic CR on temporal alterations in circulating insulin, insulin-like growth factor I/somatomedin C, insulin-like growth factor II/multiplication-stimulating activity, and epidermal growth factor levels at 0, 1, 3, 5, 11, and 20 weeks in carcinogen- and vehicle-treated animals. Tumor incidence and multiplicity were markedly inhibited (P less than 0.05) with increasing CR. Fasting serum insulin-like growth factor I/somatomedin C levels exhibited a significant acute decline with CR at 1 and 3 weeks, but were comparable to AL-fed controls throughout the remainder of the 5-month study, despite continued differences in weight gain between AL and CR rats. Levels of insulin-like growth factor II/multiplication-stimulating activity exhibited no discernible pattern in relation to CR. Serum insulin levels showed age-dependent increases, but were affected by increasing CR at all time points. Insulin levels were significantly (P less than 0.05) reduced in 40% CR rats from 3 weeks onward compared to controls, while 25% CR resulted in nonsignificant (P less than 0.07) reductions throughout the study. No significant differences in growth factor levels were observed between 7,12-dimethylbenz(a)anthracene- and vehicle-treated rats. Circulating epidermal growth factor was not detectable in any treatment group regardless of the nature or duration of the dietary regimen, time of blood collection, or subsequent tumor-bearing status. These data suggest that decreased serum insulin-like growth factor I/somatomedin C and insulin levels with CR and their complex interactions in vivo may play a role in the inhibition of mammary tumor promotion by CR.

Growth factor binding to 7,12-dimethylbenz(a)anthracene-induced mammary tumors from rats subject to chronic caloric restriction.

Caloric restriction (CR) inhibits tumorigenesis in rodents. To understand the basis for this effect the binding of insulin, insulin-like growth factor I/somatomedin C (IGF-I/Sm-C), insulin-like growth factor II/multiplication stimulating activity (IGF-II/MSA), and epidermal growth factor were examined to membrane preparations of 7,12-dimethylbenz(a)anthracene-induced mammary adenocarcinomas and several normal tissues from female Sprague-Dawley rats. Animals were fed ad libitum (AL) or 25% and 40% calorically restricted diets. Large, palpable (LP) and small, less than or equal to 100 mg, nonpalpable (SNP) tumors were evaluated. Growth factor binding to tumors was differentially affected by CR. IGF-I/Sm-C binding was comparable for AL-LP, AL-SNP, and 25% CR-LP tumors, but elevated in 25% CR-SNP tumors. Scatchard analysis revealed high and low affinity IGF-I/Sm-C binding sites, with AL-SNP and 25% CR-SNP tumors exhibiting similar levels of high affinity sites and at a greater concentration than AL-LP and 25% CR-LP tumors. Insulin binding to mammary tumors was low, i.e., 8- to 13-fold lower than IGF-I/Sm-C binding. The 25% CR-LP and SNP tumors bound 2- to 5-fold more insulin than corresponding AL-LP and SNP tumors. Binding of IGF-II/MSA to these tumor preparations was high, approximately 11- to 25-fold greater than insulin binding, and was unaffected by CR or tumor size. The binding of epidermal growth factor was not detected in any tumor preparations. Receptor binding studies were confirmed with covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Normal tissues exhibited tissue- and growth factor-specific alterations in binding with host CR. Thus, alterations in growth factor binding were not tumor specific, but were less pronounced than in mammary tumors. These findings suggest alterations in IGF-I/Sm-C and insulin binding properties to tumors in relation to CR and tumor size may contribute, in part, to the inhibitory effects of CR on tumorigenesis.

Biochemical alterations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors from rats subjected to caloric restriction.

Caloric restriction reduces the incidence and progression of a broad spectrum of neoplastic diseases, yet little is known about the biochemical and molecular mechanisms involved. Profiles of enzyme activities of importance in cellular energy utilization were examined in 7,12-dimethylbenz[a]anthracene-induced (DMBA) mammary adenocarcinomas from rats fed ad libitum or calorically restricted diets. The diets provided equal nutrients except for fewer carbohydrate-derived calories; graded caloric restriction was 10, 20, 30 and 40%. The specific activities of hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme and fructose-1,6-bisphosphatase were all elevated to varying degrees in both large palpable and small, non-palpable tumors from calorically restricted hosts compared to activities in tumors from ad libitum-fed rats. Phosphofructokinase activity was increased in palpable tumors from calorically restricted hosts but markedly reduced in non-palpable tumors. These results suggest adaptive or compensatory alterations in tumor enzyme profiles in response to the altered nutritional state of the host.

Gallstone formation in hamsters: effect of varying animal and vegetable protein levels.

The lithogenic diet routinely used for production of gallstones in hamsters contains 20% casein. It has previously been shown that replacement of casein by soy protein significantly decreases gallstone formation. In this study hamsters were fed a lithogenic diet containing casein (C), soy isolate (S), C/S 3:1, C/S 1:1, and C/S 1:3. The percentages of hamsters with gallstones on these five diets were: 44, 12, 38, 23, and 15. Biliary cholesterol levels and lithogenic index both decreased significantly with increasing levels of soy protein. Dilution of casein with soy protein progressively decreases lithogenicity.

Fatty acids and colon cancer in experimental models.

Experimental models have several advantages in the study of colon cancer. They can be used to tightly control diet, examine putative intermediate markers, test hypotheses about mechanisms of carcinogenesis, and quantify development of tumors in a short time. Dietary issues that have been studied in animal models but are unresolved include the concept of the effects of total fat compared with energy intake, composition of the basal diet, linoleic acid requirements, and interactions of fat with other nutrients. Intermediate markers that have been probed in animal or in vitro studies include cytokinetics, aberrant crypt foci, eicosanoids and hydroxyoctadecadienoic acids, ornithine decarboxylase, tyrosine kinase, protein kinase C, and gene expression. Colon cancer is studied in animals primarily with use of chemicals that are relatively specific inducers of these tumors, but transplantable models and transgenic animals are also used. Total dietary fat is generally thought to affect colon tumorigenesis, but there does not appear to be any specific fatty acid that promotes the development of colon cancer. Several studies indicate that n-3 fatty acids from marine sources alter a variety of biological intermediates and inhibit colonic tumorigenesis; this is probably mediated via the eicosanoid pathway. Although there are undoubtedly multiple cellular changes elicited by certain fatty acids, our current knowledge of this area suggests that specific fatty acid metabolites or their targets are the likely mediators in this sequence.

Influence of vegetable protein on gallstone formation in hamsters.

The lithogenic diet generally used for production of gallstones in hamsters contains 20% casein. In four separate experiments the 20% casein diet was compared with one containing 20% soy protein. All other components were kept constant. Two of the four experiments were of 45-day duration, one of 70 days and one of 100 days. When results of all four experiments were averaged, 57.5 +/- 3.6% of hamsters fed casein exhibited cholesterol gallstones whereas only 14.0 +/- 1.3% of soy protein-fed hamsters had gallstones. Thus, soy protein has a significant inhibitory effect on gallstone formation in hamsters. When soy protein was fed to hamsters with preestablished gallstones there was evidence of gallstone dissolution.

Effect of soy protein and casein intake on intestinal absorption and lymphatic transport of cholesterol and oleic acid.

Rats were fed for 4 wk on defined diets containing either casein or soy as the protein source, or diets in which the lysine/arginine ratios were modified by addition of arginine to casein, and lysine to the soy diet. During this period, weight gains and food intakes were comparable in the four dietary groups. Animals were subjected to cannulation of the left thoracic lymphatic duct, and after an overnight fast, were given a single intragastric dose of a lipid emulsion containing oleic acid and cholesterol. The overall 24-h recoveries of cholesterol and fatty acid in lymph were similar in the four groups, as were the distribution of lipids among the major lipid fractions and lipoprotein classes of thoracic duct lymph. However, analysis of timed lymph collections indicated that absorption of lipids was more rapid in casein-fed rats than in those fed soy protein. Furthermore, addition of arginine to the casein diet resulted in a slowed rate of lipid absorption, and addition of lysine to the soy diet markedly increased the rate of lipid absorption.

Influence of whole or skim milk on cholesterol metabolism in rats.

Male Wistar rats were fed commercial ration and given whole milk, skim milk, or water to drink. After 3 weeks the control group showed the greatest weight gain. Rats given whole milk had the smallest livers. Serum cholesterol levels were significantly lower in rats fed either whole or skim milk, but other serum lipids were unaffected. Liver triglyceride levels were lowest in the rats on skim milk. Activities of hepatic fatty acid synthetase, hydroxymethyl-glutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase were similar in the two milk-fed groups and considerably lower than in the controls.

An improved method for oxidation of chromium(III) oxide-containing fecal samples by using sodium peroxide fusion.

A safer method of oxidation of Cr2O3-containing fecal samples from transit-time studies was developed using sodium peroxide to replace perchloric acid as the oxidizing agent. The percentage recovery of Cr2O3 with this method was compared with that of perchloric acid method for samples containing quantities of fecal ash and Cr2O3 typical of those from rodent transit-time studies. Both methods gave relatively constant percentage recoveries for Cr2O3 contents from 0.4 to 10 mg. Over this range, mean (+/- SD) percentage recoveries of Cr2O3 for sodium peroxide fusion and the perchloric acid method were 75.5 +/- 4.3 and 89.9 +/- 2.5, respectively. As long as percentage recovery is constant, the transit time as determined by calculation of the time of 80% excretion of the total recovered Cr2O3 is not affected. Sodium peroxide fusion provides a useful and safer alternative to perchloric acid oxidation in transit-time studies using Cr2O3 as a nonabsorbable marker.

Flordipine, a calcium channel blocker, which does not influence lipidemia or atherosclerosis in cholesterol-fed rabbits.

Data relating to the effects of calcium channel blockers on experimental atherosclerosis in rabbits are inconsistent with most studies finding no effect on either serum lipids or atherosclerosis. We have administered flordipine (5, 15 or 45 mg/kg/day) for 10 weeks to rabbits fed 1% cholesterol and 4% corn oil. At no level of treatment was there an effect on serum or liver lipids or on aortic sudanophilia.

Experimental atherosclerosis in rabbits fed cholesterol-free diets.

Rabbits were fed a semipurified, cholesterol-free atherogenic diet containing 40% sucrose, 25% casein, 14% fat, 15% fiber, 5% salt mix and 1% vitamin mix. The fats were corn oil (CO), palm kernel oil (PO), cocoa butter (CB), and coconut oil (CNO). The rabbits were bled at 3, 6, and 9 months and killed at 9 months. Serum lipids of rabbits fed CO were unaffected. Serum cholesterol levels (mg/dl) at 9 months were: CO -- 64; PO -- 436; CB -- 220; and CNO -- 474. HDL-cholesterol (%) was: CO -- 37; PO -- 8.6; CB -- 25.1; and CNO -- 7.0. Average atherosclerosis (arch + thoracic/2) was: CO -- 0.15; PO -- 1.28; CB -- 0.53; and CNO -- 1.60. Cocoa butter (iodine value 33) is significantly less cholesterolemic and atherogenic than palm oil (iodine value 17) or coconut oil (iodine value 6). The difference between the atherogenic effects of cocoa butter and palm oil may lie in the fact that about half of the fatty acids of palm oil are C 16 or shorter, whereas 76% of the fatty acids of cocoa butter are C 18 or longer.

Experimental atherosclerosis in rabbits fed cholesterol-free diets. Part 12. Comparison of peanut and olive oils.

The atherogenicity of peanut oil is well established as is the fact that the structure of the component triglycerides of peanut oil influences its atherogenicity. This study was carried out to determine if the relative saturation of peanut oil was partly responsible for the observed effects. Rabbits were fed a semipurified, cholesterol-free diet containing 14% of North American peanut oil (iodine value, 100), South American peanut oil (iodine value, 110) or olive oil (iodine value, 83) for 8 months. Rabbits fed olive oil exhibited higher levels of serum and liver lipids than did the two peanut oil-fed groups but significantly lower levels of aortic atherosclerosis. The findings confirm earlier observations that the structure of a fat can have an affect on its atherosclerogenic potential that is independent of its level of unsaturation.

Increased atherosclerosis in rabbits immunized with endothelial cells.

Rabbits maintained on normal ration or cholesterol-supplemented diet were immunized with homogenates of endothelial cells grown in cultures that were derived from either bovine or human aorta. Minimal aortic lesions were found in all groups of rabbits fed regular diet; microscopically, differences were seen that manifested as medial lesions in controls and intimal lesions in animals immunized with endothelial cells. Aortic atherosclerosis was significantly increased in the immunized, cholesterol-fed animals over that in controls. This difference was more pronounced in the abdominal aorta where the area with lesions in immunized rabbits was 4-7 times that of controls; atherosclerosis of the thoracic aorta was 2.5-5 times greater in the immunized animals (P less than 0.001 for both segments). Increased atherosclerosis was observed despite a significant reduction in plasma cholesterol in the immunized rabbits (770 +/- 119 mg/dl) compared to controls (1595 +/- 225 mg/dl) (P less than 0.001). Immunization with endothelial cells elicited strong cell-mediated and humoral responses as determined by dermal delayed hypersensitivity and solid-phase immunoradiometric tests, respectively. Cross-reactivity in both assays was found against human and bovine cells. Enhancement of atherosclerosis appears to depend not on induction of immune complexes but on specific antibodies and cell-mediated reactions.

Experimental atherosclerosis in rabbits fed cholesterol-free diets. 13. Interaction of proteins and fat.

The atherogenic and cholesterolemic effects of animal protein vis-a-vis plant protein are well documented. Virtually all the studies were carried out using diets high in saturated fat, such as coconut oil. In order to determine if the same effects were seen with less saturated fat, we have compared atherogenic effects of an animal protein (casein) with those of a plant protein (soybean protein isolate) fed with partially hydrogenated soybean oil (PHS) (iodine value 72) or soybean oil (iodine value 134) as part of a cholesterol-free semipurified diet. After 6 months only rabbits fed casein-PHS exhibited elevated levels of plasma and liver cholesterol and triglycerides and atherosclerosis. Rabbits fed soy protein-PHS had slightly higher plasma cholesterol and triglycerides than did those fed soy protein and soybean oil, but values in both groups were in the normal range. The different effects of animal and plant protein on lipidemia and atherosclerosis can be influenced by dietary fat and appear to be dependent on fat saturation.

Experimental atherosclerosis in rabbits fed cholesterol-free diets. Part 9. Beef protein and textured vegetable protein.

Rabbits were fed a semipurified diet containing 40% sucrose, 25% protein, 15% fiber and 14% tallow. The proteins fed were beef (B), textured vegetable protein (TVP) and casein (C). One diet contained beef-TVP (1 : 1) and in another a soy carbohydrate fraction, spent flakes, was added to the diet; it provided 2.2% protein and 10.5% fiber. TVP provided 6.4% soluble carbohydrate and 10.5% fiber. The diets were fed from 8 months. Diets containing beef protein or casein gave significantly higher serum cholesterol levels and atherosclerosis and significantly lower serum HDL-cholesterol than did the other 3 diets. The beef-TVP (1 : 1) diet gave low serum cholesterol (67% below beef) levels and atherosclerosis (47% below beef). This effect is probably due to the protein. The diet containing spent flakes gave low serum cholesterol levels (44% below beef) and atherosclerosis (45% below beef). This effect is attributed to the different fiber. The lowest serum cholesterol levels and least severe atherosclerosis were observed in the rabbits fed TVP.