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Effect of selenium on azoxymethane-induced intestinal cancer in rats fed high fat diet
343
Cancer Letters, 12 (1981) 343-348 o Eisevier/North-Holland Scientific Publishers Ltd.
EFFECT OF SELENIUM ON AZOXYMETHANE-INDUCED CANCER IN RATS FED HIGH FAT DIET
BARBARA
INTESTINAL
K. SOULLIER, PAULETTE S. WILSON and NORMAN D. NIGRO
Department of Surgery, Matilda R. Wilson Cancer Research School of Medicine, Detroit, Michigan 48201 (U.S.A.)
Unit, Wayne State University
(Received 24 December 1980) (Accepted 20 January 1981)
SUMMARY
The effects of selenium supplementation on azoxymethane-induced intestinal cancer were studied in male Sprague- Dawley rats given 8 weekly injections of azoxymethane (8 mg/kg body wt), and fed a 30% beef fat diet. Selenium-supplemented groups received 8 ppm H,SeO, in drinking water. Blood selenium levels of supplemented rats increased rapidly the first 9 weeks of the experiment, followed by a plateau significantly higher than that for non-selenium controls. There was a significant increase in liver and intestinal selenium levels in supplemented groups. The average number of intestinal tumors was 6.5 in the control group, and 3.1 in the selenium-supplemented group. There was a significant reduction in tumor incidence in the proximal half of the colon of selenium-treated rats. There was also increased concentration of tissue selenium in the proximal half of the colon of these rats.
INTRODUCTION
The role of selenium in nutrition has been studied for many years. The element is widely distributed in nature, small amounts being present in the water, soil and in our food. The amount, however, varies in different geographic areas. In regions where levels are high, animals have developed selenium toxicity. Consequently, much of the early studies involved its toxicology, and there is no doubt that the ingestion of excessive amounts is toxic to both animals and humans [ 81. However, it has been shown to be Add&s all correspondence to: Barbara K. Soullier, c/o Norman D. Nigro, M.D., Wayne State University School of Medicine, Department of Surgery, 540 East Canfield Avenue, Detroit, MI 48201.
344
an essential trace element. The suggested intake for adult humans is about 50-200 pg, according to the 1980 edition of Recommended Dietary Allowances [3]. Selenium has also been linked to cancer. Initially, studies were reported suggesting that it is a carcinogen in animals. The evidence, however, is scant and inconclusive. More recently, epidemiological data indicates that selenium may inhibit cancer [ 71. In addition, several investigators have shown that the administration of selenium in the drinking water of animals inhibits several types of cancer, including bowel cancer. For example, Jacobs et al. [6] showed that the colon tumor incidence in rats treated with 1,2-dimethylhydrazine (DMH) was reduced by the addition of 4 ppm selenium to the drinking water. Previous experiments on the inhibitory effect of selenium were done on animals fed a normal fat diet. A high fat diet is known to double the number of tumors in rats treated with DMH or its metabolites [ 21. Consequently, if a high fat diet were given, cancer inhibition in this system of increased carcinogenic challenge would indicate that selenium has a substantial inhibitory effect. The purpose of this experiment was to evaluate the inhibitory effect of selenium on intestinal cancer in rats fed to a high fat diet and injected with azoxymethane (AOM). MATERIALS AND METHODS
AOM was obtained from Ash-Stevens Co., Detroit, MI, and was prepared as an aqueous solution for injection. Dextrose was purchased from a bakery supply house. Beef fat was donated by the Belmont Packing Co. and was rendered in the laboratory. Other dietary components were obtained from Bio-Serv, Inc., Frenchtown, NJ. To ensure consistent selenium intake, the same lot of each component was used throughout the experiment. Selenium as H,SeO, (99%) was purchased from the Ventron Corp., Alfa Products, Danvers, MA, and it was prepared as a l-g/l stock solution, neutralized with NaOH, .and added to double distilled deionized water to a final concentration of 8 mg H,SeO,/l solution. 2,3-Diamino-naphthalene monohydrochloride (DAN, 99%), was obtained from Aldrich Chemical Co., Inc., Milwaukee, WI. All other chemicals used were reagent grade. A Turner Model 110 fluorometer equipped with a No. 7-60 (365 nm) primary filter, No. 2A-12 (510 nm) secondary filter, Wratten N.D. 1.00 (10%) and Wratten N.D. 2.00 (1%) filters was used for selenium determinations. Male Sprague-Dawley rats weighing lOO-125g (6 weeks old) were purchased from Spartan Research Animals Inc., Haslett, MI. Animals were caged individually, diets and water were given ad libitum, body weights and water consumptions were recorded weekly, and food consumptions were determined every 4 weeks. All animals were fed the 30% fat semisynthetic diet presented in Table 1, which was prepared weekly in the laboratory. Eight parts per million H,SeO, (4.9 ppm Se) was added to the double distilled deionized drinking water.
345 TABLE
1
ANIMALS FED 30% FAT SEMI-SYNTHETIC
DIET
Component
Weight (%)
Total calories (%)
Protein= Salt mixb Vitamin mixC
33 7 2
26.7 -
Beef fat Dextrose Cellulose
30 23 5
54.7 18.6 -
a Vitamin-free casein: Bio-Serv No. 11319. b Salt mix USP XIV: Bio-Serv No. 20232, containing per kg salt mix: 7.8 mg cupric sulfate, 1.53 g ferric ammonium citrate, 20 mg magnesium sulfate, 9.2 mg ammonium aluminum sulfate, 4.1 mg potassium iodide, 51 mg sodium fluoride, 68.8 g calcium carbonate, 308.3 g calcium citrate, 112.8 g calcium biphosphate, 35.2 g magnesium carbonate, 38.3 g magnesium sulfate, 124.7 g potassium chloride, 218.8 g dibasic potassium phosphate, 77.1 g sodium chloride. c Vitamin Mix: Bio-Serv No. 20315, containing per kg of mixture: 4.5 g Vitamin A (200,000 U/g), 0.25 g Vitamin D (400,000 U/g), 5.0 ga!-tocopherol acetate, 5.0 g inositol, 45.0 g ascorbic acid, 158.25 g choline dihydrogen citrate, 2.25 g menadione, 5.0 g p-aminobenzoic acid, 4.5 g niacin, 1.0 g riboflavin, 1.0 g pyridoxine hydrochloride, 1.0 g thiamine hydrochloride, 3.0 g calcium pantothenate, 20 mg biotin, 90 mg folic acid, 1.35 mg Vitamin B,,, 765.24 g sucrose to make 1 kg,
Three groups of 15 animals each were fed the 30% fat diet. Two groups received S.C. injections of AOM (8mg/kg body wt) for 8 weeks while the third group received sterile water injections. One group (A) receiving AOM was given double distilled, deionized drinking water, while the second AOM group (B) and the non-AOM group (C) received 8 ppm H,SeO, throughout the experiment. Venous blood samples were collected in heparinized tubes from the retro-orbital sinus of 8 animals per group every other week. These samples were frozen until selenium determinations were made. After 25 weeks all animals were killed, necropsies were performed, and all tissues were examined for tumors. The number, size, and location of intestinal tumors were recorded. Tumor distributions between the proximal and distal halves of both the small and large intestine were tabulated. Histologic preparations were made of liver, normal intestinal tissue, and tumors. Tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. The remaining intestine and the livers were frozen until selenium determinations were made. In order to determine the uptake of selenium by specific intestinal segments, 2 groups of 15 animals each were fed the 30% fat, diet. One group was given distilled, deionized drinking water and the second group was given 8 ppm H,Se03 in the drinking water. The rats were killed after 9 weeks, and intestinal tissue from 8 animals was frozen until selenium determinations were made. The small and large intestines were divided into proximal and distal halves for selenium determinations.
346
0”“““““” 2 4
6
8
10
12 14
16 18 20
22 24 26
WEEKS
Fig. 1. Average blood selenium levels of 8 animals/group vs. time. There was a rapid increase in blood selenium levels of supplemented rats during the first 9 weeks followed by a significantly higher (P < 0.0005) plateau region for those groups compared to non-
supplemented rats. Selenium concentrations were determined by the fluorometric procedure of Watkinson [9]. Samples of liver (0.25 g), intestine (0.5 g) and blood (1.0 g) were predigested with nitric acid overnight at room temperature before digesting with perchloric acid, All samples were run in triplicate. All data were analyzed with Student’s t-test [ 11. RESULTS
There was essentially no difference in weight gain, food consumption, or water consumption attributable either to AOM treatment or selenium supplementation throughout the course of this study. Figure 1 shows an initial rapid increase in selenium blood levels of supplemented rats (Groups B and C) during the first 9 weeks, followed by a plateau region significantly higher (P d 0.0005) than that for the non-selenium controls (Group A). Similarly, selenium levels in the liver and intestinal tract increased by a factor of 2 with selenium supplementation (Table 2). TABLE 2 AVERAGE
SELENIUM
LEVELS
(fig/g tissue + S.D.)
Group
Treatment
Liver
Small intestine
Large intestine
A B C
AOM AOM + Se Se
0.45 + 0.14 0.85 * 0.14 0.84 * 0.23
0.22 f 0.07 0.43 + 0.07 0.42 2 0.12
0.34 f 0.07 0.62 + 0.09 0.56 2 0.07
347
The gross appearance of the liver in all animals in this experiment was normal. Histologic sections were made from the liver of 6 rats in each group injected with AOM.-There were minimal degrees of fatty degeneration and focal necrosis in the liver of 2 rats from each group. All others were normal. Since these findings in the 2 groups were essentially the same, there was no evidence of toxicity in the liver of rats given the selenium supplement. All animals given AOM injections developed intestinal tumors, while there were none in the non-carcinogen treated animals given the selenium supplement. Unfortunately, 16 rats died prematurely, the result of an improper anesthetic used during bleeding. This left fewer animals than planned for the analysis of intestinal tumor formation. There were 9 rats in Group A (AOM only) and 10 in Group B (AOM + Se). The average number of intestinal tumors was 6.5 in Group A and 3.1 in Group B. The distribution of tumors in the various segments of the intestine is shown in Fig. 2. There were fewer tumors in the small intestine in Group B compared to Group A, but the difference was not significant. However, there was a marked reduction in the number of tumors in the proximal half of the colon in Group B compared to Group A. This reduction was statistically significant (P < 0.005). The tumors varied in size from 2 mm to 1.4 cm in greatest diameter and all were adenocarcinoma on histologic examination. There was no significant difference between the 2 groups (A and B) receiving the carcinogen in either the size or the degree of anaplasia. However, the number of tumors in the proximal half of the colon in the selenium-supplem,ented group was too small to make any valid histological comparison. The segment-specific selenium uptake study showed a significantly
0
GROUPA-AOM
q GROUPB-AOM,Se
Pmxkml
usml
PlWil-Ml
SMALL BOWEL LARGE BOWEL
Fig. 2. Tumor frequency (average no. of tumors/rat) and distribution for each AOM treated group. There was a significant reduction (P < 0.005) in the number of tumors in the proximal, half of the colon in the selenium-supplemented group.
348
increased (P < 0.05) concentration of tissue selenium in the proximal half of the colon for supplemented rats (proximal 0.35 pg Se/g tissue, distal 0.27 pg/g). Control rats had an even distribution of selenium throughout the colon (proximal 0.20 pg/g, distal 0.21 pg/g). DISCUSSION
The total carcinogenic challenge (initiator + promoter) used in these experiments was considerably higher than in previous studies. Even with this increased challenge, the selenium supplement (8 ppm H,SeO,) had a significant inhibitory effect on a colonic carcinogenesis. Interestingly, the reduction was entirely in the proximal half of the colon. This segment-specific reduction in tumors correlates well with the observed preferential uptake of supplemental selenium by that part of the colon. Thus, the higher concentration of selenium is indicated to be the source of inhibition; however, further study is necessary to determine the specific form of selenium and the pathway involved. Frequently proposed mechanisms in such systems are alterations in carcinogen metabolism [ 51 and protection against oxidative damage through the action of the selenoenzyme glutathione peroxidase [ 41. The latter mechanism is of particular interest in this study because of the increased lipid concentration resulting from the 30% fat diet. ACKNOWLEDGEMENTS
We acknowledge the excellent technical assistance of Marc Duchane. also thank Marilyn Reinbold for her skillful secretarial assistance.
We
REFERENCES 1 Beyer. W. (1968) Handbookof tables for probability and statistics. 2nd edn. Chemical Rubber Co., Cleveland, Ohio. Bull, A.W., Soullier, B.K., Wilson, P.W., Hayden, M.T. and Nigro, N.D. (1979) Promotion of azoxymethane-induced intestinal cancer by high fat diets in rats. Cancer Res., 39,4956-4959. Food and Nutrition Board, Committee on Dietary Allowances. (1980) Recommended dietary allowances, 9th edn., pp. 162-163. National Academy of Sciences, Washington, DC. Griffin, A.C. (1979) Role of selenium in the chemoprevention of cancer. Adv. Cancer Res., 29, 419-442. Jacobs, M.M. (1977) Inhibitory effects of selenium on 1,2dimethylhydrazine and methylazoxymethanol colon carcinogenesis. Cancer, 40, 2557-2564. Jacobs, M.M., Birger, J. and Griffin, A.C. (1977) Inhibitory effects of selenium on 1,2-dimethylhydrazine and methylazoxymethanol acetate induction of colon tumors. Cancer Letters, 2, 133-138. Schrauzer, G.N. (1977) Trace elements, nutrition and cancer: perspectives of prevention. In: Inorganic and Nutritional Aspects of Cancer, pp. 323-344. Editor: G.N. Schrauzer. Plenum Press, New York. Shapiro, J.R. (1973) Selenium and carcinogenesis: a review. Ann. N.Y. Acad. Sci., 204,215-219. Watkinson, J.H. (1966) Fluorometric determination of selenium in biological material with 2,3diaminonaphthalene. Anal. Chem., 38,92-97.
Cancer Letters, 12 (1981) 343-348 o Eisevier/North-Holland Scientific Publishers Ltd.
EFFECT OF SELENIUM ON AZOXYMETHANE-INDUCED CANCER IN RATS FED HIGH FAT DIET
BARBARA
INTESTINAL
K. SOULLIER, PAULETTE S. WILSON and NORMAN D. NIGRO
Department of Surgery, Matilda R. Wilson Cancer Research School of Medicine, Detroit, Michigan 48201 (U.S.A.)
Unit, Wayne State University
(Received 24 December 1980) (Accepted 20 January 1981)
SUMMARY
The effects of selenium supplementation on azoxymethane-induced intestinal cancer were studied in male Sprague- Dawley rats given 8 weekly injections of azoxymethane (8 mg/kg body wt), and fed a 30% beef fat diet. Selenium-supplemented groups received 8 ppm H,SeO, in drinking water. Blood selenium levels of supplemented rats increased rapidly the first 9 weeks of the experiment, followed by a plateau significantly higher than that for non-selenium controls. There was a significant increase in liver and intestinal selenium levels in supplemented groups. The average number of intestinal tumors was 6.5 in the control group, and 3.1 in the selenium-supplemented group. There was a significant reduction in tumor incidence in the proximal half of the colon of selenium-treated rats. There was also increased concentration of tissue selenium in the proximal half of the colon of these rats.
INTRODUCTION
The role of selenium in nutrition has been studied for many years. The element is widely distributed in nature, small amounts being present in the water, soil and in our food. The amount, however, varies in different geographic areas. In regions where levels are high, animals have developed selenium toxicity. Consequently, much of the early studies involved its toxicology, and there is no doubt that the ingestion of excessive amounts is toxic to both animals and humans [ 81. However, it has been shown to be Add&s all correspondence to: Barbara K. Soullier, c/o Norman D. Nigro, M.D., Wayne State University School of Medicine, Department of Surgery, 540 East Canfield Avenue, Detroit, MI 48201.
344
an essential trace element. The suggested intake for adult humans is about 50-200 pg, according to the 1980 edition of Recommended Dietary Allowances [3]. Selenium has also been linked to cancer. Initially, studies were reported suggesting that it is a carcinogen in animals. The evidence, however, is scant and inconclusive. More recently, epidemiological data indicates that selenium may inhibit cancer [ 71. In addition, several investigators have shown that the administration of selenium in the drinking water of animals inhibits several types of cancer, including bowel cancer. For example, Jacobs et al. [6] showed that the colon tumor incidence in rats treated with 1,2-dimethylhydrazine (DMH) was reduced by the addition of 4 ppm selenium to the drinking water. Previous experiments on the inhibitory effect of selenium were done on animals fed a normal fat diet. A high fat diet is known to double the number of tumors in rats treated with DMH or its metabolites [ 21. Consequently, if a high fat diet were given, cancer inhibition in this system of increased carcinogenic challenge would indicate that selenium has a substantial inhibitory effect. The purpose of this experiment was to evaluate the inhibitory effect of selenium on intestinal cancer in rats fed to a high fat diet and injected with azoxymethane (AOM). MATERIALS AND METHODS
AOM was obtained from Ash-Stevens Co., Detroit, MI, and was prepared as an aqueous solution for injection. Dextrose was purchased from a bakery supply house. Beef fat was donated by the Belmont Packing Co. and was rendered in the laboratory. Other dietary components were obtained from Bio-Serv, Inc., Frenchtown, NJ. To ensure consistent selenium intake, the same lot of each component was used throughout the experiment. Selenium as H,SeO, (99%) was purchased from the Ventron Corp., Alfa Products, Danvers, MA, and it was prepared as a l-g/l stock solution, neutralized with NaOH, .and added to double distilled deionized water to a final concentration of 8 mg H,SeO,/l solution. 2,3-Diamino-naphthalene monohydrochloride (DAN, 99%), was obtained from Aldrich Chemical Co., Inc., Milwaukee, WI. All other chemicals used were reagent grade. A Turner Model 110 fluorometer equipped with a No. 7-60 (365 nm) primary filter, No. 2A-12 (510 nm) secondary filter, Wratten N.D. 1.00 (10%) and Wratten N.D. 2.00 (1%) filters was used for selenium determinations. Male Sprague-Dawley rats weighing lOO-125g (6 weeks old) were purchased from Spartan Research Animals Inc., Haslett, MI. Animals were caged individually, diets and water were given ad libitum, body weights and water consumptions were recorded weekly, and food consumptions were determined every 4 weeks. All animals were fed the 30% fat semisynthetic diet presented in Table 1, which was prepared weekly in the laboratory. Eight parts per million H,SeO, (4.9 ppm Se) was added to the double distilled deionized drinking water.
345 TABLE
1
ANIMALS FED 30% FAT SEMI-SYNTHETIC
DIET
Component
Weight (%)
Total calories (%)
Protein= Salt mixb Vitamin mixC
33 7 2
26.7 -
Beef fat Dextrose Cellulose
30 23 5
54.7 18.6 -
a Vitamin-free casein: Bio-Serv No. 11319. b Salt mix USP XIV: Bio-Serv No. 20232, containing per kg salt mix: 7.8 mg cupric sulfate, 1.53 g ferric ammonium citrate, 20 mg magnesium sulfate, 9.2 mg ammonium aluminum sulfate, 4.1 mg potassium iodide, 51 mg sodium fluoride, 68.8 g calcium carbonate, 308.3 g calcium citrate, 112.8 g calcium biphosphate, 35.2 g magnesium carbonate, 38.3 g magnesium sulfate, 124.7 g potassium chloride, 218.8 g dibasic potassium phosphate, 77.1 g sodium chloride. c Vitamin Mix: Bio-Serv No. 20315, containing per kg of mixture: 4.5 g Vitamin A (200,000 U/g), 0.25 g Vitamin D (400,000 U/g), 5.0 ga!-tocopherol acetate, 5.0 g inositol, 45.0 g ascorbic acid, 158.25 g choline dihydrogen citrate, 2.25 g menadione, 5.0 g p-aminobenzoic acid, 4.5 g niacin, 1.0 g riboflavin, 1.0 g pyridoxine hydrochloride, 1.0 g thiamine hydrochloride, 3.0 g calcium pantothenate, 20 mg biotin, 90 mg folic acid, 1.35 mg Vitamin B,,, 765.24 g sucrose to make 1 kg,
Three groups of 15 animals each were fed the 30% fat diet. Two groups received S.C. injections of AOM (8mg/kg body wt) for 8 weeks while the third group received sterile water injections. One group (A) receiving AOM was given double distilled, deionized drinking water, while the second AOM group (B) and the non-AOM group (C) received 8 ppm H,SeO, throughout the experiment. Venous blood samples were collected in heparinized tubes from the retro-orbital sinus of 8 animals per group every other week. These samples were frozen until selenium determinations were made. After 25 weeks all animals were killed, necropsies were performed, and all tissues were examined for tumors. The number, size, and location of intestinal tumors were recorded. Tumor distributions between the proximal and distal halves of both the small and large intestine were tabulated. Histologic preparations were made of liver, normal intestinal tissue, and tumors. Tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. The remaining intestine and the livers were frozen until selenium determinations were made. In order to determine the uptake of selenium by specific intestinal segments, 2 groups of 15 animals each were fed the 30% fat, diet. One group was given distilled, deionized drinking water and the second group was given 8 ppm H,Se03 in the drinking water. The rats were killed after 9 weeks, and intestinal tissue from 8 animals was frozen until selenium determinations were made. The small and large intestines were divided into proximal and distal halves for selenium determinations.
346
0”“““““” 2 4
6
8
10
12 14
16 18 20
22 24 26
WEEKS
Fig. 1. Average blood selenium levels of 8 animals/group vs. time. There was a rapid increase in blood selenium levels of supplemented rats during the first 9 weeks followed by a significantly higher (P < 0.0005) plateau region for those groups compared to non-
supplemented rats. Selenium concentrations were determined by the fluorometric procedure of Watkinson [9]. Samples of liver (0.25 g), intestine (0.5 g) and blood (1.0 g) were predigested with nitric acid overnight at room temperature before digesting with perchloric acid, All samples were run in triplicate. All data were analyzed with Student’s t-test [ 11. RESULTS
There was essentially no difference in weight gain, food consumption, or water consumption attributable either to AOM treatment or selenium supplementation throughout the course of this study. Figure 1 shows an initial rapid increase in selenium blood levels of supplemented rats (Groups B and C) during the first 9 weeks, followed by a plateau region significantly higher (P d 0.0005) than that for the non-selenium controls (Group A). Similarly, selenium levels in the liver and intestinal tract increased by a factor of 2 with selenium supplementation (Table 2). TABLE 2 AVERAGE
SELENIUM
LEVELS
(fig/g tissue + S.D.)
Group
Treatment
Liver
Small intestine
Large intestine
A B C
AOM AOM + Se Se
0.45 + 0.14 0.85 * 0.14 0.84 * 0.23
0.22 f 0.07 0.43 + 0.07 0.42 2 0.12
0.34 f 0.07 0.62 + 0.09 0.56 2 0.07
347
The gross appearance of the liver in all animals in this experiment was normal. Histologic sections were made from the liver of 6 rats in each group injected with AOM.-There were minimal degrees of fatty degeneration and focal necrosis in the liver of 2 rats from each group. All others were normal. Since these findings in the 2 groups were essentially the same, there was no evidence of toxicity in the liver of rats given the selenium supplement. All animals given AOM injections developed intestinal tumors, while there were none in the non-carcinogen treated animals given the selenium supplement. Unfortunately, 16 rats died prematurely, the result of an improper anesthetic used during bleeding. This left fewer animals than planned for the analysis of intestinal tumor formation. There were 9 rats in Group A (AOM only) and 10 in Group B (AOM + Se). The average number of intestinal tumors was 6.5 in Group A and 3.1 in Group B. The distribution of tumors in the various segments of the intestine is shown in Fig. 2. There were fewer tumors in the small intestine in Group B compared to Group A, but the difference was not significant. However, there was a marked reduction in the number of tumors in the proximal half of the colon in Group B compared to Group A. This reduction was statistically significant (P < 0.005). The tumors varied in size from 2 mm to 1.4 cm in greatest diameter and all were adenocarcinoma on histologic examination. There was no significant difference between the 2 groups (A and B) receiving the carcinogen in either the size or the degree of anaplasia. However, the number of tumors in the proximal half of the colon in the selenium-supplem,ented group was too small to make any valid histological comparison. The segment-specific selenium uptake study showed a significantly
0
GROUPA-AOM
q GROUPB-AOM,Se
Pmxkml
usml
PlWil-Ml
SMALL BOWEL LARGE BOWEL
Fig. 2. Tumor frequency (average no. of tumors/rat) and distribution for each AOM treated group. There was a significant reduction (P < 0.005) in the number of tumors in the proximal, half of the colon in the selenium-supplemented group.
348
increased (P < 0.05) concentration of tissue selenium in the proximal half of the colon for supplemented rats (proximal 0.35 pg Se/g tissue, distal 0.27 pg/g). Control rats had an even distribution of selenium throughout the colon (proximal 0.20 pg/g, distal 0.21 pg/g). DISCUSSION
The total carcinogenic challenge (initiator + promoter) used in these experiments was considerably higher than in previous studies. Even with this increased challenge, the selenium supplement (8 ppm H,SeO,) had a significant inhibitory effect on a colonic carcinogenesis. Interestingly, the reduction was entirely in the proximal half of the colon. This segment-specific reduction in tumors correlates well with the observed preferential uptake of supplemental selenium by that part of the colon. Thus, the higher concentration of selenium is indicated to be the source of inhibition; however, further study is necessary to determine the specific form of selenium and the pathway involved. Frequently proposed mechanisms in such systems are alterations in carcinogen metabolism [ 51 and protection against oxidative damage through the action of the selenoenzyme glutathione peroxidase [ 41. The latter mechanism is of particular interest in this study because of the increased lipid concentration resulting from the 30% fat diet. ACKNOWLEDGEMENTS
We acknowledge the excellent technical assistance of Marc Duchane. also thank Marilyn Reinbold for her skillful secretarial assistance.
We
REFERENCES 1 Beyer. W. (1968) Handbookof tables for probability and statistics. 2nd edn. Chemical Rubber Co., Cleveland, Ohio. Bull, A.W., Soullier, B.K., Wilson, P.W., Hayden, M.T. and Nigro, N.D. (1979) Promotion of azoxymethane-induced intestinal cancer by high fat diets in rats. Cancer Res., 39,4956-4959. Food and Nutrition Board, Committee on Dietary Allowances. (1980) Recommended dietary allowances, 9th edn., pp. 162-163. National Academy of Sciences, Washington, DC. Griffin, A.C. (1979) Role of selenium in the chemoprevention of cancer. Adv. Cancer Res., 29, 419-442. Jacobs, M.M. (1977) Inhibitory effects of selenium on 1,2dimethylhydrazine and methylazoxymethanol colon carcinogenesis. Cancer, 40, 2557-2564. Jacobs, M.M., Birger, J. and Griffin, A.C. (1977) Inhibitory effects of selenium on 1,2-dimethylhydrazine and methylazoxymethanol acetate induction of colon tumors. Cancer Letters, 2, 133-138. Schrauzer, G.N. (1977) Trace elements, nutrition and cancer: perspectives of prevention. In: Inorganic and Nutritional Aspects of Cancer, pp. 323-344. Editor: G.N. Schrauzer. Plenum Press, New York. Shapiro, J.R. (1973) Selenium and carcinogenesis: a review. Ann. N.Y. Acad. Sci., 204,215-219. Watkinson, J.H. (1966) Fluorometric determination of selenium in biological material with 2,3diaminonaphthalene. Anal. Chem., 38,92-97.