Dimethyihydrazine-induced colon tumors in rats fed diets containing beef fat or corn oil with and without wheat bran1 2

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1 Dimethyihydrazine-induced colon tumors in rats fed diets containing beef fat or corn oil with and without wheat bran1 2 Robert B. Wilson, David P. Hutcheson, and Linda Wideman ABSTRACT Male rats of the Sprague-Dawley strain were fed semisynthetic diets containing either 20% beef fat or corn oil with and without 20% wheat bran. Half of the animals received four weekly doses and the other half received eight weekly doses of dimethylhydrazine, 30 mg/kg, by intragastric intubation. The percentage of rats of the colon of all types was significantly higher in animals fed no bran than in those fed bran. Likewise, the percentage of rats with polypoid neoplasms of the colon was higher in rats fed no bran, but there was no significant difference in the percentage of rats with malignant tumors of the colon with respect to the feeding of bran. No significant differences were found between rats fed corn oil and those fed beef fat with respect to either the incidence or the kinds of colon tumors. Malignant tumors of the colon, causing death, occurred earlier in rats fed corn oil as compared to those fed beef fat. The percentge of rats of the colon and the numbers of tumors per tumor-bearing rat were significantly increased in rats given eight doses of dimethylhydrazine or compared to those given four. Am. J. Clin. Nut,. 30: , The relative lack of fiber (1, 2) and cereals (3) and the abundance of animal fat and protein (4-7) in the American diet have been cited as important factors associated with the high incidence of cancer of the colon in this country. Studies using animal models of cancer of the colon have shown that increasing the amount of dietary fat results in a higher incidence of tumors, but no clear differences have been reported relative to the role of plant versus animal fats (8-10). In one study (11), dietary cellulose failed to alter carcinogenesis in an experimental model, but the effects of other types of dietary fiber have not been reported. The purposes of the present study were to compare the time of onset, tumor incidence, and type of tumors induced by a chemical carcinogen, sym-dimethyihydrazine (DMH), in rats fed semisynthetic diets containing either corn oil or beef fat, with and without wheat bran as a source of fiber. Male rats of the Sprague-Dawley strain (176) weighing approximately 380 g, were assigned, in equal numbers, to one of four groups. The rats had been previously housed in our facilities and fed laboratory chow since weaning. They were housed individually in an airconditioned, humidity-controlled room and provided with 12 hr of light and 12 hr of darkness each day. Water and food were provided free-choice. The rats were fed one of four experimental diets throughout the study (Table 1). They were given either 4 or 8 weekly doses of DMH (30 mg/kg) by gastric intubation. The study, therefore, consisted of four dietary groups and two subgroups (doses) within each dietary group for a total of eight groups. The amounts of dietary ingredients were adjusted to balance the amount of protein in wheat bran and cholesterol in beef fat so that all diets contained approximately the same amounts of fat, cholesterol, and protein. The vitamin and mineral content of the wheat bran were not considered in the dietary calculations. The addition or deletion of the nonprotein portion of the wheat bran was countered by deleting or adding sucrose (Table 1). The diets were mixed every two weeks and stored at 5 C. Fresh food was offered daily. The rats were observed daily and weighed every 2 weeks. After the fourth dose of DMH, six rats from each dietary group were placed in metabolic cages and, after a 4-day period of adjustment, food intake and fecal weight were determined daily for the next 3 days. All surviving animals were killed 8 weeks after the first rat died from complications related to cancer of the Materials and methods From the Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA (R. B. W.) and Sinclair Research Farm, University of Missouri, Columbia, Missouri (D. P. H. and L. W.). 2 Supported in part by a grant from the National Livestock and Meat Board. 176 TheAmerican Journal of Clinical Nutrition 30: FEBRUARY 1977, pp Printed in U.S.A.

2 DIMETHYLHYDRAZINE-INDUCED COLON TUMORS IN RATS 177 colon. The digestive tube was opened from the anterior portion of the esophagus to the anus. The location of tumors was established by measuring from either the pylorus or the anus to the tumor locus. The tumors were described and measured in-the-gross and then fixed in neutral-buffered-formalin. Histological sections were processed from the liver and kidneys of each animal as well as from organs and tissues containing what appeared to be metastatic tumors or tumors of tissues other than those of the digestive tract. Paraffinembedded tissue sections were stained with either hematoxylin and eosin or Periodic acid-schiff reagent and alcian blue. The intestinal tumors were classified according to the depth of invasion of the intestine, and histological type as described by Ward (12). Data were expressed as the percentage of tumor-bearing rats per group and the number of tumors per tumor-bearing rats. Differences among groups were tested by the square method. Results Food intake was similar among the groups and was approximately 20 g/day. Differences in body weights were not significant at any time (Table 2). By casual observation, it was noted that the amount of feces expelled by the rats consuming bran was several times that of the animals not consuming bran, an observation substantiated by the 3- day fecal collection trial (Table 3). TABLE 1 Dietary ingredients Beaf fat basal Beef fat bran Corn oil basal Cm oil bran Wheat bran Casein Sucrose Beef fat Corn oil Vitamin mix#{176} Mineral mixb Cholesterol Total a Vitamin mix to supply the following vitamins per kilogram of complete diet: A, 2000 IU; D, 100 IU; E, 35 mg; K, 0.05 mg; ca pantothenate, 8 mg; nacin, 15 mg; riboflavin, 2.5 mg; thiamin, 1.25 mg; pyridoxine, 7 mg; B,2,.005 mg; folic acid, 0.5 mg; biotin, 300 g; inositol, 1 gm; para-aminobenzoic acid, 100 mg; choline chloride, 750 mg. b Mineral mix to supply the following minerals per kilogram of complete diet: Dicalcium phosphate dihydrate, 21.5 gm; sodium chloride, 1.27 gm; potassium carbonate, gm; magnesium sulfate, 1.98 gm; cupric sulfate, mg; pitassium iodide, 0.20 mg; ferous sulfate heptahydrate, mg; zinc carbonate, mg; sodium selenate, 0.10 mg; manganous sulfate, mg. TABLE 2 Body weights Group (N = 44)C Start of dosing End of dosing First tumors Beeffatbasal 376 ± ± ± 75 Beeffatbran 392 ± ± ± 60 Cornoilbasal 379 ± ± ± 69 Corn oil bran 380 ± ± ± 80 a Subgroups (4 doses and 8 doses) not significantly different in weight b (Mean ± SD g). TABLE 3 Three-day fecal collection Six animals/ groups Initial weight Beef fat basal 502 ( ) Beef fat bran 511 ( ) Corn oil basal 500 ( ) Corn oil bran 517 ( ) a Mean (range). g Diet intake 19.4 ( ) 20.1 (13-25) 18.6 ( ) 17.8 (11-23) giday Feces weight 1.6 ( ) 4.1 ( ) 1.3 ( ) 3.7 ( ) During the 5th month, four rats died that had been fed corn oil. All had mucinous adenocarcinomas of the colon that had metastasized to the peritoneum, pancreas, liver and, in two cases, the lungs. No such deaths had occurred in rats fed beef fat. The difference in the deaths between the corn oil and beef fat groups was significant (P < 0.05) at this time. During the 6th month, a few spontaneous deaths from complications of colon tumors occurred in the beef fat group so that at the time the survivors were killed, midway during the 7th month, the difference in death loss between the two groups was not significant. Description ofcolon tumors Most tumors were found in the colon; none was noted in the cecum and only a few were observed in the small intestine. Grossly, the tumors of the colon were either polypoid or plaque-like growths. Tumors were observed throughout the colon, but were more commonly found in the descending portion. Hemorrhages of various severity and intusseptions associated with colon tumors were observed in approximately 20% of rats. Metastatic growths from tumors of the colon were oh-

3 178 WILSON ET AL. served in nine animals. Metastatic sites included regional lymph nodes, peritoneum, pancreas, liver, spleen, and lungs. Histologically, tumors of the colon were classified as polypoid neoplasms, adenocarcinomas, or mucinous adenocarcinomas. Polyploid neoplasms grew into the lumen of the colon and developed stalks of variable lengths and thicknesses. Epithelium covering these tumors was deeply basophilic. The stalks contained connective tissue and blood vessels. Neoplastic cells did not invade deeper than the tunica muscularis. Adenocarcinomas grew into the wall of the colon up to the serosa. The invasion was accompanied by a marked stromal reaction. Mucinous adenocarcinomas appeared to arise most commonly in lymphoid follicles. They consisted of clusters of epithelial cells, many of which appeared as signet rings and were Periodic acid-schiff positive, separated by a thin connective tissue stroma. These tumors proved to be the most malignant and metastasized to the peritoneum, regional lymph nodes, pancreas, spleen, and lungs. Metastatic lesions provoked a marked stromal reaction with considerable necrosis, except in the lungs. Description of other tumors and lesions Tumors of the small intestine were few and limited to the duodenum; most were TABLE 4 Total tumors in rats given DMH Group found near the entrance of the common bile duct. Histologically, these tumors were classified as were the colon tumors. Invasion by adenocarcinomas and mucinous adenocarcinomas was limited to the serosa; none metastasized. Squamous cell carcinomas of the ear canal were locally invasive, but they did not metastasize to other tissues. Various degrees of megalocytosis was seen in the livers of almost all rats that had been given eight doses of the carcinogen. Numbers of tumors The percentage of tumor-bearing rats and the numbers of tumors per tumor-bearing rat are given in Table 4. The percentage of rats of the colon was significantly higher in rats fed no bran as compared to those fed bran and in rats given eight doses of DMH as compared to those given four, but the difference in tumor mcidence between rats fed corn oil and beef fat was not significant. Of rats fed wheat bran 63% had no colon tumors at all while only 3 1 % of rats not fed wheat bran were free of tumors. However, the two rats with the most colon tumors (10 and 11) were fed bran, thereby increasing the average numbers of tumors per tumor-bearing rat. The numbers of tumors per tumor-bearing rat Colon Small intestine Ear duct rats rats No. tumors rats 1. Beef fat, nobran 68}a Beeffat,bran Corn oil, no bran Corn oil, bran Ia. Beef fat, no bran a. Beef fat, bran a.Cornoil,nobran 90} a. Corn oil, bran Overall comparison Beef fat Corn oil Bran Nobran 52}s } 1.3k iJ a S = significant differe nce P <0.001.

4 DIMEThYLHYDRAZINE-INDUCED COLON TUMORS IN RATS 179 was significantly higher in those given eight doses as compared to those given four. The difference in the percentage of rats with colon tumors was accounted for by the difference in the number of polypoid neoplasms (Tables 5 and 6); differences in the percentages of rats with obviously malignant tumors were not significant. The incidence of tumors of the small intestine and the ear canal was small in all groups and differences were not significant with respect to either dietary treatment of dose of DMH, except that the incidence of tumors of the ear canal TABLE 5 Polyploid neoplasms of the colon Group Beef fat, no bran 661 Beef fat, bran Corn oil, no bran Corn oil,bran Beef fat, no bran Beef fat, bran Corn oil, no bran Corn oil, bran 48 J S Overall comparisons Beef fat Corn oil Bran 2.9 No bran 69J 2.0 4Doses 41} 65 a 5 = significant difference P < TABLE 6 Malignant colon tumors Group rat Beef fat, no bran Beef fat, bran Corn oil, no bran Corn oil, bran 26 Beef fat, no bran 20 Beef fat, bran 28 Corn oil, no bran Cron oil, bran 30 Overall comparisons Beef fat 25 Cornoil Bran 28 No bran rat was significantly higher in rats given eight doses of DMH as compared to those given four (Table 4). Discussion The cause of tumors of the colon in man in unknown, but chemical carcinogens from the environment or produced within the body from foods are important candidates. The role of dietary ingredients as either primary or secondary factors in cancer of the colon is of current interest (8). In the present study, the addition of wheat bran to the diet greatly increased the daily excretion of feces and provided a measure of protection against polypoid neoplasm of the colon, but not against more malignant tumors. Since bran contains some vitamins and minerals in addition to fiber and carbohydrate, its effects may not be attributable to fiber alone. We did not control for the total composition of wheat bran in this study. Ward et al. (1 1) observed fewer tumors of the small intestine induced by azoxymethane in rats fed 20 or 40% cellulose, but tumors of the colon were not affected by dietary cellulose. Corn oil enhanced the growth of experimental mammary tumors (13). On the other hand, it has been postulated that high intake of saturated fat, particularly beef fat, might be an important causitive factor in the genesis of cancer of the colon in man (4-7). In the present study, the incidence and total number of various tumors were not influenced significantly by the kind of dietary fat. Twice as many malignant tumors of the colon were observed in rats fed corn oil, without bran and given eight doses of DMH than any other group, but differences did not reach statistical significance. It is noteworthy, however, that four rats in this group died from complications of malignant tumors of the colon 4 weeks before similar deaths occurred in other groups. Nigro et al. (14) reported an increased incidence of tumors of the colon induced by azoxymethane in rats fed 35% beef fat mixed with laboratory chow as compared to animals given the carcinogen and fed chow alone. However, the addition of fat to laboratory chow, dilutes other food factors, making interpretation difficult. Large amounts of dietary fat of any kind is known

5 180 WILSON ET AL. to enhance experimental carcinogenesis in a number of models. For example, Reddy et a!. (8) observed more tumors of the colon in rats treated with either 20% lard or corn oil as compared to either fat at the 5% level. More tumors were found in rats fed corn oil than lard, but differences were not statistically significant. Rogers and Newberne (9) found no differences in carcinogenesis in rats given DMH and fed diets containing either beef fat or corn oil, however, carcinogenesis was enhanced by high-fat diets contaming marginal amounts of lipotropes (10). Presumably, the low-lipotrope diets altered hepatic drug metabolism. These investigators also found that vitamin A deficiency increased the number of colon tumors in rats treated with aflatoxin B, but the affect in rats given DMH was inconclusive (15). Two doses of DMH (4 and 8) were used because in a preliminary experiment (R. B. Wilson et al. unpublished data) it was found that about 75% of animals fed laboratory chow and given eight doses developed tumors of the colon whereas only 20% of rats similarly fed and given four doses developed tumors. It was reasoned that the lowerdosed groups might facilitate detection of any carcinogenic enhancing effect of a diet. The incidence and total number of colon tumors was significantly increased in the groups given 8 doses and compared to those given only four; however, the incidence and total number of tumors in the latter groups were higher than had been observed previously in rats fed chow. The large amount of fat (20%) in our semisynthetic diets might have enhanced carcinogenesis as previously noted. Neither the incidence nor the total number of malignant tumors of the colon was significantly influenced by the kind of dietary fat, the presence or absence of dietary wheat bran, or the number of doses. Since the overall incidence of these tumors was low, significant differences may have emerged had the number of animals in each group been larger. It is also possible, that with additional time, a number of tumors that were classified as benign might have developed a more invasive character, or our classification criteria might have been unduly conservative, but care was taken to insure that the same histological criteria were applied to tumors of all groups. The results of this study taken together with the results of others of the effects of dietary alterations on carcinogenesis in experimental models of cancer of the colon may be summarized as follows: 1 ) Increasing the amount of dietary fat enhanced carcinogenesis, but no significant differences were observed between animal or plant fats fed at the same level. 2) Vitamin A deficiency resulted in inconsistent enhancement of carcinogenesis. 3) Diets low in lipotropes enhanced carcinogenesis. 4) Dietary cellulose failed to alter the mcidence of colon tumors, but the addition of wheat bran to the diet significantly decreased the number of benign colon tumors. U References 1. BuRiurr, D. P. Epidemiology of cancer of the colon and rectum. Cancer 28: 3, BURKITT, D. P. Large bowel cancer: an epidemiologic jigsaw puzzle. J. NatI. Cancer Inst. 54: 3, IRVING, D., AND B. S. DRASAR. Fibre and cancer of the colon. Brit. J. Cancer. 28: 462, HAENSZEL, W., J. W. BERG, M. Sai, M. KURI- HARA AND F. B. LOCKE. Large bowel cancer in Hawaiian Japanese. J. Natl. Cancer Inst. 51: 1765, WYNDER, E. L. The epidemiology of large bowel cancer. Cancer Res. 35: 3388, WYNDER, E. L., AND B. S. REDDY. Dietary fat and colon cancer. J. Natl. Cancer Inst. 54: 7, ApjismoNG, B., AND R. DOLL. Environmental factors and cancer incidence and morality in different countries, with special reference to dietary practices. Intern. J. Cancer 15: 617, REDDY, B. S.,T. NARISAWA, R. MARONPOT, J. H. WEISBURGER AND E. L. WYNDER. Animal models for the study of dietary factors and cancer of the large bowel. Cancer Res. 35: 3421, RooEas, A. E. AND P. M. NEWBERNE. Dietary effects on chemical carcinogenesis in animal models for colon and liver tumors. Cancer Res. 35: 3427, ROGERS, A. E., AND P. M. NEWBERNE. Dietary enhancement of intestinal carcinogenesis by dimethylhydrazine in rats. Nature 246: 491, WARD, J. M., R. S. YAMAMOTO AND J. H. Wms- BURGER. Brief communication: cellulose, dietary bulk and azoxymethane-induced intestinal cancer. J. Natl. Cancer Inst. 51: 713, WARD, J. M. Morphogenesis of chemically induced neoplasms of the colon and small intestine in rats. Lab. Invest. 30: 505, GAMMAL, E. B., K. K. CARROLL AND E. R. PLUN-

6 DIMETHYLHYDRAZINE-INDUCED COLON TUMORS IN RATS 181 KErr. Effects of dietary fat on mammary carcinogenesis by 7, 12-dimethylbenzanthracene in rats. Cancer Res. 27: 1737, NIGRO, N. D., D. V. SINGH, R. L. CAMPBELL AND M. S. PAK. Effect of dietary beef fat on intestinal tumor formation by azoxymethane in rats. J. Natl. Cancer Inst. 54: 439, ROGERS, A. E., B. J. HERNDON AND P. M. NEW- BERNE. Induction by dimethyhydrazine of intestinal carcinomas in normal rats and rats fed high or low levels of vitamin A. Cancer Res. 33: 1003, 1973.