REPORTS

Influence of Dietary Calcium and Vitamin D on Diet-Induced Epithelial Cell Hyperproliferation in Mice

Lexun Xue, Martin Lipkin, Harold Newmark, Jiarmin Wang

Affiliations of authors: L. Xue, Memorial Sloan-Kettering Cancer Center and Strang Cancer Prevention Center, New York, NY, and Cell Biology Laboratory, Laboratory Center of Medical Sciences, Henan Medical University, Zhengzhou, China; M. Lipkin, H. Newmark, Memorial Sloan-Kettering Cancer Center and Strang Cancer Prevention Center; J. Wang, Cell Biology Laboratory, Laboratory Center of Medical Sciences, Henan Medical University.

Correspondence to: Martin Lipkin, M.D., Strang Cancer Research Laboratory at The Rockefeller University, 1230 York Ave., New York, NY 10021.


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Previous epidemiologic and laboratory studies, including some from our own laboratory, have suggested that a high-fat diet increases risk of cancer development in the pancreas, prostate, colon, and breast and that carcinogenesis in some of these organs may be influenced by alterations in dietary calcium and vitamin D. In this study, we sought to investigate the effect of added dietary calcium or vitamin D on the development of epithelial cell hyperproliferation induced by a Western-style diet in the exocrine pancreas, prostate, and mammary gland of mice. METHODS: Four-week-old C57BL/6J mice were given either a control diet (American Institute of Nutrition [AIN]-76A), a Western-style diet (containing reduced calcium and vitamin D and the fat level of the average human Western diet), or a putative chemopreventive diet (a Western-style diet with the addition of dietary calcium and vitamin D). Nine weeks after dietary intervention, osmotic pumps were implanted in the mice to provide 3 days of bromodeoxyuridine (BrdU) infusion. All P values are two-sided. RESULTS: Mice on the Western-style diet had statistically significant increases in BrdU-labeling indices of epithelial cells in the interlobular (P = .015) and intralobular (P = .012) ducts and centroacinar cells (P = .001) of the pancreatic duct system, the dorsal lobe of the prostate (P = .045), and the terminal ducts of the mammary gland (P = .032), compared with mice in the respective control diet groups. Adding dietary calcium and vitamin D markedly suppressed the Western-style diet-induced hyperproliferation of epithelial cells in those tissues (P = .001-.033). CONCLUSIONS: This study confirms previous findings that a Western-style diet produces hyperproliferation of epithelial cells in several organs and that the changes can be prevented by increasing dietary calcium and vitamin D alone.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Breast cancer in females and prostatic cancer in males are among the most frequently diagnosed cancers in the United States and some West European countries (1-5). Moreover, pancreatic cancer remains one of the most important human cancers because reliable methods of early disease detection and effective treatment are lacking (6). Although an understanding of the causes and mode of carcinogenesis of these diseases is still lacking, several epidemiologic and experimental studies have suggested that high fat consumption and decreased calcium and vitamin D intake are associated with carcinogenesis of the pancreas (1,7-12), prostate (4,13-20), and mammary gland (1,17,21-32).

Complete reduction or elimination of human exposure to environmental factors such as high-fat diets is inherently difficult to implement. Studies (33-39) have begun to evaluate a possible role for increased dietary calcium and vitamin D in reducing the risk of colonic and mammary cancers, even in the presence of a high-fat diet. Furthermore, we (40,41) found that decreased dietary calcium and vitamin D in a high-fat diet induced epithelial cell hyperproliferation in the pancreas and prostate gland, which suggests a possible role for increased dietary calcium and vitamin D in the chemoprevention of pancreatic and prostatic cancers.

In this study, we (a) again administrated a Western-style diet to mice and studied cell proliferation in the exocrine pancreas, prostate, and mammary gland and (b) investigated the inhibitory effects of calcium and vitamin D added to the Western-style diet on epithelial cell hyperproliferation in those organs.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Diets

The diets were based on a semipurified rat and mouse diet developed by the American Institute of Nutrition (AIN), i.e., AIN-76A (42). The control diet was the complete form of this diet, which meets known nutritional requirements for rats and mice and supports their growth and development. As described in our previous studies (40,41,43-45), the following modified forms of the AIN-76A diet were used as experimental diets. Western-style diet (WSD 1) contained increased levels of fat and phosphate and reduced calcium and vitamin D content, simulating levels found in human Western diets. The putative chemopreventive diet (WSD 2) had the same amount of fat and phosphate as WSD 1 but contained increased levels of calcium and vitamin D. The calcium in WSD 2 was raised to 7 mg/g feed (1.5 mg/kilocalories [kcal]), slightly higher than that in the control AIN-76A diet (1.4 mg/kcal). At 1.5 mg/kcal, this would approximate 3000 mg in a 2000-kcal daily human diet. The level of vitamin D in WSD 2 was raised to 2.3 IU/g feed (0.5 IU/kcal), slightly higher than that in the AIN-76A diet. At 0.5 IU/kcal, this would approximate 1000 IU in a 2000-kcal human diet. The higher levels of calcium and vitamin D are similar to some recommendations for the prevention of osteoporosis in humans (46). Details on the diets used in this study are given in Table 1.Go


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Table 1. Diet composition*

 
Animals and Treatment

Three-week-old C57BL/6J mice of both sexes were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed at the animal facility of Memorial Sloan-Kettering Cancer Center, which is accredited by the American Association for Accreditation of Laboratory Animal Care. Animal care was provided in accordance with guidelines outlined in Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 86-23, 1985). Mice of the same sex were housed four or five per cage in standard wire-top cages with sawdust bedding. They were kept on a 12-hour light-dark cycle, at 50% (±5%) relative humidity, in a temperature-controlled (approximately 23 °C) room. After 1 week of acclimatization on the AIN-76A control diet, all of the animals were randomly assigned by weight to one of three groups (control, WSD 1, and WSD 2 groups) and fed one of the three diets ad libitum. Each diet group consisted of six to seven male and six to seven female mice. They had free access to food and water throughout the experiment. Mice were inspected daily for general health and weighed weekly before and during the study.

After 9 weeks of dietary intervention, all the animals received bromodeoxyuridine (BrdU) (1.44 mg per mouse), which was administrated continuously for 72 hours by implanted Alzet osmotic pumps (Model 1003D; Alza Corporation, Palo Alto, CA) as described previously (40,41). For this infusion, mice were anesthetized with intraperitoneal injections of sodium pentobarbital at 40 mg/kg of body weight. The osmotic pumps were subcutaneously implanted over the dorsal thoracolumbar areas in anesthetized mice, and incisions were closed with nylon sutures by use of aseptic technique. These pumps began continuous delivery at a constant rate of 1 µL/hour beginning 4 hours after pump implantation. All the animals were killed by cervical dislocation 76 hours after pump implantation. At necropsy, the pancreas, prostate, and mammary gland were removed, respectively, and fixed in an 80% ethanol solution for 24 hours and then in a 95% ethanol solution. Tissues were embedded in paraffin, and tissue sections were cut 4 µm thick and stained either with hematoxylin-eosin for histopathologic examination or immunohistochemically for BrdU. The slides were analyzed without knowledge of the source of the specimens.

Morphometry

(a) Pancreas. The histology of the mouse exocrine pancreas has been described previously (40). The exocrine pancreas is a compound acinar gland consisting of a branching duct system and clusters of glandular acini. The duct system includes interlobular and intralobular ducts and centroacinar (centroductular) cells. The acinus consists of a single layer of pyramid-shaped cells with the narrow apical ends bordering the lumen.

(b) Prostate. The mouse prostate is composed of paired anterior (the rodent anterior lobe is sometimes referred to as coagulating gland), dorsal, and ventral lobes that—unlike those of man—have not merged into one compact anatomic structure (41,47,48). Two of these lobes, the anterior and dorsal, are homologous to zones in the human prostate. One mouse lobe, the ventral lobe, does not appear to have a human homologue (48).

(c) Mammary gland. As described in our previous study (45), the mouse mammary gland consists of three types of ducts (i.e., terminal, intermediate, and large ducts).

Immunohistochemical Analysis

Proliferating cells were labeled immunohistochemically by use of BrdU as described previously (40,41). Briefly, tissue sections were deparaffinized in xylene, treated with methanol containing 0.6% hydrogen peroxide, denatured with 2N hydrochloric acid, incubated with 10% normal horse serum, and reacted with 1 : 1000 BrdU monoclonal antibody (Becton Dickinson, Mountain View, CA). Subsequently, the tissue sections were reacted with 1% biotinylated horse anti-mouse immunoglobulin G and then with avidin-biotin peroxidase complex (Vectastain ABC peroxidase kit; Vector Laboratories, Burlingame, CA). Finally, the sections were developed with a 0.03% 3'3-diaminobenzidine tetrahydrochloride solution (Sigma Chemical Co., St. Louis, MO), counterstained with hematoxylin, dehydrated, cleared, and mounted under coverslips with crystal mount.

BrdU-positive cells were identified by brown to black pigment over their nuclei and were counted by microscopy. Epithelial cell proliferation was expressed as a labeling index, which was calculated as the ratio of the number of BrdU-labeled cells to the total number of epithelial cells counted.

For the pancreas, 1 x 106 µm2 of pancreatic tissue was examined under 400x magnification for each mouse. The total number of ductal or acinar epithelial cells and the total number of BrdU-labeled cells were counted, respectively, in each of the three types of ducts or acini.

On the prostate slides, 1000 acinar epithelial cells per mouse from 10 or more acini were counted, and the BrdU-labeled cells in each of the three lobes were counted for each mouse.

In the mammary gland, under light microscopy with 400x magnification, 200 fields per animal were examined. The total number of mammary ductal epithelial cells and the total number of BrdU-labeled ductal epithelial cells were counted, respectively, in each microscopic field for each of the three types of ducts.

Statistical Analysis

The data were analyzed by two-way analysis of variance (49). Summary statistics were expressed as mean values. (Error bars give 95% confidence intervals.) All P values for the differences are two-sided and considered statistically significant for P<.05.


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tolerance for Diets and Body Weights

At the beginning of the experiment, there were no significant differences in body weights between any of the diet groups for mice of the same sex, and such differences were not statistically significant after 9 weeks of dietary intervention. All mice tolerated each of the diets well, and no mice died during the 9-week experimental period.

Studies of Morphometry and Immunohistochemistry

Pancreas. As shown in Fig. 1,Go the control group was similar to both the WSD 1 and WSD 2 groups in the number of ducts (interlobular and intralobular ducts and centroacinar cells) and acini of exocrine pancreatic tissue assayed per mouse. This result further demonstrated that neither WSD 1 nor WSD 2 influenced the growth of ducts or acini during the 9-week feeding period in this study.



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Fig. 1. Effects of Western-style diet (WSD) 1—which is high in fat and phosphate content but reduced in calcium and vitamin D levels compared with the American Institute of Nutrition (AIN)-76A control diet (CD)—and WSD 2—which is identical to WSD 1, except for increased levels of calcium and vitamin D—on multiplicity of ducts and acini (top) and on cell proliferation (bottom) in the pancreas of mice. Values are means (error bars give 95% confidence intervals) and reflect ductal and acinar multiplicity rate and epithelial cell proliferation rate (bromodeoxyuridine-labeling index [BrdU-LI]) in the ductal systems (interlobular and intralobular ducts and centroacinar cells) and acini of the respective diet groups after 9 weeks of diet administration. Two-tailed P values (*P<.05 and **P<.01) indicate statistically significant differences between the WSD 1 group and the control (CD) or WSD 2 groups, respectively, by two-way analysis of variance.

 
The effects of feeding the three types of diets on epithelial cell proliferation in the pancreatic duct system and acini are as follows:

(a) Interlobular ducts—The BrdU-labeling index of the WSD 1 group was markedly elevated compared with that of both the control (P = .015) and the WSD 2 (P = .019) groups. The BrdU-labeling index of the WSD 2 group was slightly higher than that of the control group, but there was no statistically significant difference between those groups.

(b) Intralobular ducts—The BrdU-labeling index of the WSD 1 group was significantly higher than that of the control (P = .012) or the WSD 2 (P = .026) group. The BrdU-labeling indices in the control and WSD 2 groups were similar.

(c) Centroacinar (centroductular) cells—A significant increase (P = .001) was present in the BrdU-labeling index of the WSD 1 group compared with that of both the control and the WSD 2 groups. There was no statistically significant difference between the control and WSD 2 groups in the BrdU-labeling indices.

The findings described here indicated that feeding the WSD 1, which contains increased fat content and low levels of calcium and vitamin D, induced epithelial cell hyperproliferation in the ductal system of the mouse exocrine pancreas (particularly in the centroacinar cells) and also that this hyperproliferative effect of the WSD 1 was prevented by dietary supplementation with calcium and vitamin D.

(d) Acini—There were no statistically significant changes between any of the three diet groups in the BrdU-labeling indices, although the BrdU-labeling index of the control group was lower than that of either the WSD 1 or the WSD 2 group (Fig. 1Go).

Prostate. The effects of feeding the WSD 1 and WSD 2 diets for 9 weeks on epithelial cell proliferation in the mouse prostate are shown in Fig. 2.Go A statistically significant increase in the BrdU-labeling index of epithelial cells in the dorsal lobe was present after feeding the animals WSD 1 for 9 weeks compared with the findings in the control (P = .045) or WSD 2 (P = .033) group; however, there were no statistically significant differences in the BrdU-labeling indices of epithelial cells in the anterior and ventral lobes between the WSD 1, WSD 2, and control groups after 9 weeks of dietary intervention. Thus, the findings further demonstrated that feeding WSD 1 to mice led to epithelial cell hyperproliferation in the dorsal lobe of the mouse prostate, a region that is homologous with the dorsal zone of the human prostate, and that dietary calcium and vitamin D suppressed the prostatic epithelial cell hyperproliferation induced by WSD 1.



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Fig. 2. Effects of Western-style diet (WSD) 1—which is high in fat and phosphate content but reduced in calcium and vitamin D levels compared with the American Institute of Nutrition (AIN)-76A control diet (CD)—and WSD 2—which is identical to WSD 1, except for increased levels of calcium and vitamin D—on proliferation of epithelial cells in the prostate of mice. Values are means (error bars give 95% confidence intervals) of bromodeoxyuridine-labeling indexes (BrdU-LIs) of epithelial cells in three types of prostate lobes. *Two-sided P<.05 indicates that the BrdU-LI of epithelial cells of the WSD 1 group was significantly elevated compared with the same measure for the CD and WSD 2 groups, respectively, by two-way analysis of variance. For each diet group, there were six or seven mice.

 
Mammary gland. Fig. 3Go summarizes the effects of feeding WSD 1 and WSD 2 on epithelial cell hyperproliferation and multiplicity (number of ducts per animal) in the mouse mammary gland. The number and the BrdU-labeling indices of the large and intermediate ducts in the WSD 1 and WSD 2 groups were similar to those in the control group at the termination of the experiment. After the 9-week feeding period, the numbers of small-terminal ducts in the control, WSD 1, and WSD 2 groups were similar, but a statistically significant increase (P = .032) was present in the BrdU-labeling index of epithelial cells in the terminal ducts of mice fed WSD 1 compared with animals fed the control or WSD 2 diet. These findings indicated that WSD 1 induced hyperproliferation of epithelial cells in terminal mammary ducts, which are a cancer-prone region of the mammary glands in both rodents and humans, and that the effect of WSD 1 was inhibited by the addition of dietary calcium and vitamin D.



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Fig. 3. Number of mammary ducts per mouse (top) and bromodeoxyuridine-labeling index (BrdU-LI) of epithelial cells in mammary ducts (bottom). Values are means (error bars give 95% confidence intervals). *Two-sided P<.05 indicates statistically significant differences between the WSD 1 group and the control (CD) and WSD 1 groups, respectively, by use of two-way analysis of variance.

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, ductal multiplicity and epithelial cell proliferation in the exocrine pancreas, prostate, and mammary glands of mice were investigated after 9 weeks of dietary intervention. We found that the BrdU-labeling indices of epithelial cells from interlobular and intralobular ducts and of centroacinar cells of the exocrine pancreas were much higher after WSD 1 feeding than after WSD 2 or control diet feeding, without statistically significant differences occurring in the number of pancreatic ducts or acini. In addition, after WSD 1 feeding, the BrdU-labeling indices of epithelial cells in the dorsal lobe of mouse prostate and in mammary terminal ducts were significantly increased compared with findings seen after control-diet or WSD 2 feeding.

Previous studies from our laboratory (43,44,50,51) have shown that a Western-style diet induced hyperproliferation, hyperplasia, and whole-crypt dysplasia (a late-stage preneoplastic lesion) in the colon and that calcium supplementation inhibited those changes. We also demonstrated that feeding mice a Western-style diet without any carcinogens induced epithelial cell hyperproliferation in the exocrine pancreas (40), prostate (41), and mammary gland (45), which suggests that administration of a diet with low levels of calcium and vitamin D together with an increased fat content might also enhance carcinogenesis in those organs, similar to what we found for the colon (43,44).

Epidemiologic investigations have suggested that low calcium levels and increased dietary fat content are associated with the increased incidences of cancers of the breast (1,17), prostate (1,4,15-20), and pancreas (1,10,12). Previous animal studies (7-9,13,14,22-27,29,31) also have supported an association of fat intake, types of fatty acids, and those cancers. In addition to decreased calcium levels, the WSD 1 used in the present study contained 20% corn oil, which is rich in omega-6 (n-6) polyunsaturated fatty acids (57%), principally linoleic acid. A previous study by us (51) found no statistically significant differences in epithelial cell proliferation between mice fed such a diet long-term or that were fed a comparable diet whose fat source was American Blend fat, which simulates the mixed lipid intake in the average U.S. diet.

It has been demonstrated that diets high in n-6 polyunsaturated fatty acids promote tumor formation at several sites—colon, breast, prostate, and pancreas. Rose and Connolly (52) reported that diets high in n-6 polyunsaturated fatty acids stimulated the growth of prostatic cancer cells. In addition, findings from animal and human epidemiologic studies (1,10-12,53) have associated high fat consumption with pancreatic carcinogenesis. For example, Birt et al. (53) found that feeding hamsters a diet containing 20% by weight of n-6 polyunsaturated fatty acids induced more pancreatic cancers than did feeding hamsters a diet containing 5% of such fats. In general, altering the levels and types of dietary fat markedly influences the development of mammary tumors in rodents (25). When animals were fed a high-fat diet, mammary tumor incidence increased sharply, and the latent period for mammary tumors was shortened as compared with that in control animals given a low or moderate level of dietary fat. Also, diets rich in n-6 polyunsaturated fatty acids, e.g., corn oil, are more efficacious in enhancing mammary tumorigenesis than diets rich in saturated fatty acids, e.g., beef tallow (29). The findings mentioned above thus suggest that dietary n-6 polyunsaturated fatty acids may well have contributed to the increased epithelial cell proliferation in the mouse pancreas, prostate, and mammary gland observed in this study and as had been noted previously in the colon (43,44).

The dietary components studied here, calcium and vitamin D, have been observed to be potent in inhibiting epithelial cell hyperplasia and hyperproliferation in the colon and mammary gland (22,33-39,50). Dietary calcium has, itself, been shown to decrease the hyperproliferation of colonic epithelium in high-fat (corn oil) diets similar to the findings with WSD 1 (44,50,54). A plausible mechanism for the antiproliferative action of dietary calcium is through its binding to bile and fatty acids (55) and through direct inhibitory action on colonic epithelial cells (35).

Other studies (56,57) have demonstrated that the active metabolite of vitamin D—1,25-dihydroxyvitamin D3 [1,25(OH)2D3]—has an important role in maintaining calcium homeostasis by mediating calcium absorption into intestinal and other cells. Active vitamin D metabolites also induce cell differentiation and inhibit proliferation in a variety of human breast cancer cells (56,57). Dietary vitamin D was shown to inhibit the growth of chemically induced rat mammary tumors (30,56). The effects of 1,25(OH)2D3 are mediated via the vitamin D receptor in mammary tissues. The results obtained in our study are consistent with the hypothesis that low levels of vitamin D contribute to the development of epithelial cell hyperproliferation in the terminal mammary ducts, possibly by reducing the absorption of calcium by the mammary cells.

Corder et al. (58) also have observed that low serum levels of 1,25(OH)2D3 were associated with an increased risk of prostatic cancer in older men. It has been shown that vitamin D receptors are present in both normal and cancerous cells of the prostate, indicating that prostate cells are target cells responding to vitamin D (59,60). Studies (59-62) also have demonstrated that 1,25(OH)2D3 inhibits the growth of normal and cancerous cells of the prostate in vitro.

Thus, a short, 9-week feeding period of the WSD 1—which provided the animals in this study with decreased levels of dietary calcium and vitamin D, coupled with increased fat content—induced hyperproliferation in the epithelial cells of the pancreas, prostate, and mammary gland. Conversely, WSD 2, which contains the same constituents as WSD 1 but contains more calcium and vitamin D than are found in WSD 1, prevented the development of hyperproliferation in epithelial cells of those organs, as had been observed previously in the colon (43).

Previous and recent studies in humans (63-65) have demonstrated that increasing the daily intake of calcium to about 1200 mg in subjects at risk for colonic neoplasia reduces the proliferative activity of colonic epithelial cells and restores markers of normal differentiation. The most recent study in humans (65) increased dietary calcium intake with a natural food source, low-fat dairy foods, confirming previous studies (34-36,63, 64) with calcium carbonate supplements that demonstrated protective effects of calcium on human colonic epithelial cells. These findings considered together suggest that further study of dietary calcium and vitamin D should be undertaken to determine their effects as chemopreventive agents against carcinogenesis of the pancreas, prostate, and breast in populations on Western diets.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received March 24, 1998; revised October 23, 1998; accepted November 3, 1998.


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