Carcinogenesis vol.12 no.11 pp.2041-2045, 1991
Phytic acid and minerals: effect on early markers of risk for mammary and colon carcinogenesis
Lilian U.Thompson and Lin Zhang Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
Introduction Epidemiological studies have shown a close relationship between colon and breast cancers (1-3) and a possibility that they have some common etiologic factors has been suggested. Certain dietary factors such as fat have been implicated as enhancing agents in both cancers (1,4,5). However, the fact that dietary fiber intake is high in vegetarians or semi-vegetarians who have lower cancer incidence than omnivores or non-vegetarians (1,2,6) indicates that dietary fiber or some substances associated with it such as inositol hexaphosphate or phytic acid (PA) may have some cancer protective effect (1,2,6,7). Previous work in our laboratory and others have shown that PA can lower the cell proliferative activity and tumor incidence and growth in the colon of rats and mice (8 -14). It is not known whether PA can also affect mammary cancer. However, because of the close relationship between the two cancers, we hypothesize 'Abbreviations: PA, inositol hexaphosphate or phytic acid; NA, nuclear aberration; IDP, intraductal proliferation; DMBA, dimethylbenzanthracene; LI, labeling index; MI, mitotic index; TEB, terminal end bud; AB, alveolar bud; TD, terminal duct. Diets: H, high-fat diet; HPA, diet H with PA; HCa, diet H with high Ca level; HFe, diet H with high Fe level; HPACA, diet H with PA and high Ca level; HPAFe, diet H with PA and high Fe level.
Materials and methods Animals In two experiments, 3-week old female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were used. The animals were housed in wire-top plastic cages maintained at 23°C with 12 h light/dark cycle. Diets and deionized water were provided ad libitum. Experimental diets All diets were based on the AIN-76 composition (15) wrth modifications for type of carbohydrate (only com starch instead of sucrose and com starch) and level of fat, minerals and PA used. Experimental diet L, was a low-fat control diet containing 5% com oil, while diet H was a high-fat control diet with 25% corn oil. Diets HCa, HFe, HPA, HPACa, and HPAFe were all high-fat (25%) diets with 1.5% total Ca (as CaHPO4), 535 p.p.m. total Fe (as FeSO4-7 H2O), 1.2% PA (as sodium phytate) alone or PA in combination with the Ca or Fe respectively. Fat, PA, Fe and Ca were added at the expense of corn starch. All diet ingredients were obtained from ICN Biochemicals (Costa Mesa, CA) except com starch (St Lawrence Starch Ltd, Mississauga, Ontario), com oil (Mazola, Toronto, Ontario), sodium phytate (Sigma Chemical Co, St Louis, MO), CaHPO4 and FeSO-7 H2O (Fisher Scientific Co., Toronto, Ontario). The control diets contained 0.5% Ca, 35 p.p.m. Fe and no PA; therefore, the experimental design essentially tested the effect of two levels (i.e. high and low) of the additives. The 1.2% PA level may be approached in a lacto-ovo-vegetarian diet (8,16). The high PA, Ca and Fe levels were chosen because they are close to the levels that have been shown individually to cause some effect on the colon or mammary ceils (8-14, 17-22) and, hence, are ideal in demonstrating interactions. A high-fat diet was used as the primary control since it is known to increase cancer risk (1,4,5). A low-fat diet was included as an additional control since it has been associated with lower risk for carcinogenesis. Experiment 1 Eighty-four female mice were randomized into seven groups, acclimatized for 1 week and then fed their special diets and deionized water for 2 weeks. Body weights were monitored during the feeding period. At the end of the feeding period, half of the animals per group were given injections of ccJchicine (1 /jg/g body wt; Sigma) and [3H]thymidine (2 fiCi/g, body wt; Amersham Corp. Arlington Heights, IL) and 2 h later were killed. The thoracic mammary glands and colon tissues were removed, stretched flat on a filter paper, fixed in 10% neutral formalin and prepared for histcJogical examination. Mammary gland and colon tissues were embedded in paraffin blocks, sectioned ( 4 - 5 jim) and transferred to slides. One set of slides was dewaxed, processed for autoradiography using standard techniques (8,17,23) and examined microscopically for number and position of labeled cells. Another set of slides was dewaxed, directly stained with H&E and examined for number of cells undergoing mitosis. The other half of the animals per group were fasted overnight and given orally a single dose of the mammary carcinogen dimethylbenzanthracene (DMBA; 100
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This study determined the effect of inositol hexaphosphate or phytic acid (PA; 1.2%), Ca (1.5%) and Fe (535 p.p.m.) alone, and PA in combination with Ca or Fe in a high-fat diet (25%) on the labeling (LI) and mitotk (MI) cell proliferation indices, nuclear aberration (NA) and intraductal proliferation (IDP) in the mammary gland, as well as the LI in colonic epithelial cells. Diet supplementation with PA alone caused reductions (P < 0.05) in the colon LI by 18%, and in the LI and NA in the total mammary gland structures of mice by 29 and 30% respectively. Supplementation with Fe or particularly Ca caused increases hi the colon LI and in the mammary LI, MI, NA and IDP but these were reduced by 25-53% (P < 0.05) in the presence of PA. These results support the hypothesis that PA may reduce the risk for both colon and mammary cancer and its effect is related to its mineral binding ability. Furthermore, significant relationships (P < 0.01) were observed between the LI and MI or NA in the total structures of the mammary gland. The number of IDPs also related (P < 0.05) to LI or NA in the terminal end bud structure of the mammary gland, suggesting that highly proliferating mammary cells, particularly in the terminal end bud structure, are of greater risk for nuclear damage and development to IDP. A significant relationship (P < 0.01) was observed between the cell proliferation in the mammary gland and that in the colon, indicating that both tissues can be influenced similarly by dietary constituents.
that a similar protective effect for mammary cancer may also be observed. The mechanisms whereby PA may affect cancer development is not clear. Nevertheless, since PA is a highly negatively charged molecule that can bind with minerals such as Fe and Ca, we also hypothesize that its effect may be partly related to its mineral binding ability. Therefore the overall objective of this study was to determine the effect of PA on early markers of risk for mammary and colon carcinogenesis, and to determine if the effect is related to the PA's mineral binding ability. Specifically, we determined the effect of PA, Ca and Fe alone, or PA in combination with Ca or Fe on (i) the cell proliferation, nuclear aberration (NA) and intraductal proliferation (IDP) in the mammary gland and (ii) cell proliferation in the colonic epithelial cell. We also correlated the cell proliferative activities in the colon and in the mammary gland.
L.U.Thompson and L.Zhang
Experiment 2 Fifty-six female mice were divided into seven diet groups, acclimatized for 1 week and fed their special diets for 2 weeks as in experiment 1. After fasting overnight, they were given orally DMBA (100 mg/kg body wt) in com oil, and then given back their same special diet for another 3 weeks. Body weight was monitored three times a week during the feeding period. At the end of the experiment, mammary glands were removed, sectioned, stained with H&E as above, and microscopically examined for IDP (25-27). When DMBA is introduced when TEBs are plentiful and actively differentiating into ABs, many TEBs do not eventually differentiate; instead they become larger and at this stage are called the IDPs (25-27). While TEBs are characterized by 3 - 6 layers of epithelial cell lining or layer, the IDP has 6—10 layers. Confluence of IDP leads to the formation of a microtumor, which in time becomes a palpable tumor. Statistical analysis The mean standard error of the mean were calculated for each diet groups. Statistical differences between groups in LI, MI, NA and IDP formation in the breast and/or colon tissues were calculated by one-way analysis of variance followed by Duncan's multiple range test. Regression analysis was done between the risk marker indices.
Table I. Effect of various diets on LJ, MI and NA in the total and different structures of the mammary gland Diet group*
Terminal end bud
L H HFe HCa HPA HPAFe HPACa
14.41 17.22 20.11 20.04 11.59 11.66 12.50
± ± ± ± ± ± ±
0.73 c 0.80 b 0.42" 0.63" 0.6^ 0.67 d 0.81 d
2.73 3.08 4.03 3.64 2.00 1.51 1.77
± ± ± ± ± ± ±
L H HFe HCa HPA HPAFe HPACa
3.17 3.51 4.71 5.19 2.80 2.95 3.19
± ± ± ± ± ± ±
0.22 b 0.47 b 0.41" 0.51' 0.11 b 0.22 b 0.10 b
0.98 1.04 1.61 2.05 0.91 0.81 0.93
± ± ± ± ± ± ±
L H HFe HCa HPA HPAFe HPACa
7.06 8.30 9.16 9.53 6.45 6.81 6.86
± ± ± ± ± ± ±
0.20 b 0.86 < b 0.64 1 0.77* 0.29 b 0.71 b 0.34 b
0.93 0.76 0.86 0.90 0.65 0.69 0.79
± ± ± ± ± ± ±
Terminal duct LI 0.61 b ' c d 0.28*'b'c 0.29* 0.56 l b
o.ird d 0.33 0.29"1
Alveolar bud
Total
1.77 1.75 2.51 2.12 1.20 1.36 0.89
± 0.38lbc ± 0.22"'b-c ± 0.45" ± 0.41" b ± 0.1 l c ±O.32 b - c ± 019bC
7.15 8.06 9.36 9.46 5.69 5.58 5.52
± ± ± ±
0.20"
0.25 0.43 0.47 0.95 0.28 0.28 0.35
± ± ± ± ± ± ±
0.07 b 0.15 b 0.14 b 0.09* 0.08 b 0.03 b 0.05 b
2.19 2.36 2.87 3.74 1.53 1.81 1.77
± ± ± ± ± ±
0.16bc 0.33bc 0.29 b 0.50" 0.09 c 0.13°
*
o.or
2.43 2.19 2.04 2.56 1.56 1.81 1.92
± 0.15lb ± 0.24 l b 0.26 b c ± 0.11" ± 0.18° ± 0.2 l b c ± 0.11 b c
3.72 4.56 4.71 4.87 3.19 3.36 3.11
± 0.13 b c 0.34 l b ± 0.41* b ± 0.42" ± 0.18° ± 0.46c ± 0.19°
O.SO1' 0.32" 0.45" O.33 c ± 0.32 c ± 0.44 c
MI 0.13 b 0.09" O.35 l b 0.50" 0.09 b 0.18 b 0.06" NA 0.07* 0.1 l " b 0.07*-b 0.1 l " b 0.04b 0.06 l b 0.03lb
•L, low fat (5%); H, high fat (25%); HPA, high fat with 1.2% PA; HFe, high fat with 535 p.p.m. Fe; HCa, high fat with 1.5% Ca; HPAFe, high fat with 1.2% PA and 535 p.p.m. Fe; HPACa, high fat with 1.2% PA and 1.5% Ca. Means within the same column with different superscripts are significantly different (P < 0.05).
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Results Experiment I No significant difference in the final body weight was observed between diet groups. While all high-fat diet groups also did not differ significantly with each other in weight gain, they had - 14% higher values (P < 0.05) than the low-fat diet group. Table I shows that high Ca or Fe supplementation of the highfat diet significantly increased the LI in the total structures of the mammary gland, while PA supplementation alone or in combination with Ca or Fe significantly decreased it. When the location of the LJ in the different mammary gland structures was examined, a similar pattern of changes were observed. However, not only was the LI value the highest in the TEB, but also more significant differences in LJ were seen in the TEB compared with in the TD and the AB structures, e.g. while the differences in LJ between the H and HFe, HCa, HPA, HPAFe or HPACa, between HFe and HPAFe and between HCa and HPACa groups were significant in the TEB, only the last four differences were significant in the TD and only the difference between HFe and HPAFe groups was significant in the AB structure. Regarding the MI, a similar trend as the LJ was seen in both the total and in the different structures of the mammary gland (Table I); thus, a significant relationship (r = 0.92; P < 0.01) was observed between the LI and MI in the total structures of the mammary gland. The NA was significantly higher in the total mammary gland structures of the high-fat and mineral-supplemented diet groups compared with all the PA-supplemented diet groups (Table I). In all groups, the NA was the highest in the TEB compared to
mg/kg body wt) in 0.2 ml com oil as established by Sharkey and Bruce (24). After 24 h, the animals were killed and their mammary glands were removed, fixed in formalin, prepared for histology, and directly stained with H&E for examination of NA as previously described (24). The labeling index (LI), indicating the number of epithelial cells undergoing DNA synthesis, was estimated as the percentage of epithelial cells labeled by tritiated thymidine, while the mhotic index (MI) was estimated as the percentage of cells undergoing mitosis. NA is the percentage of epithelial cells with nuclear aberration which can be seen in the mammary tissues after DMBA treatment. The LI, MI and NA in the total and different structures of the mammary gland, i.e. terminal end bud (TEB), alveolar bud (AB) and terminal duct (TD), were determined. The nature and appearance of these structures have been well described (25-27). The AB is formed from the normal differentiation of the TEB structure. A large number of the TEBs do not differentiate, become progressively smaller and form the finger-shaped TD structures.
Ptiytk add and minerals
Table n. Effect of various diets on the LI in the colon epithelial cells and the IDP in the mammary gland of mice Diet groups'
Colon LI (experiment 1)
L H HFe HCa HPA HPAFe HPACa
10.15 10.98 13.92 13.49 9.03 9.11 9.22
± ± ± ± ± ± ±
IDP (experiment 2)
0.28 bc 0 31 b O.571 0.32* 0.10° 0.39* 0.34c
1.50 2.88 4.50 5.38 3.00 3.38 3.14
± ± ± ± ± ± ±
0.06C 0.35 b c 0.87* b 0.73* 0.53 b c 0.68 b c 0.52 b c
•For symbols, see Table I. Means within the same column with different superscripts are significantly different (P < 0.05)
I
between the LI in the total structure of the mammary gland (Table I) and the LI in the colon epithelial cells (Table II). Experiment 2 No significant differences in final body weight and weight gain were seen between diet groups. The high-fat group did not differ in number of IDPs compared with all the other groups, with the exception of the HCa group which had a significantly higher number of IDPs (Table H). PA supplementation, however, significantly decreased the number of IDPs in the group fed the high Ca-supplemented diet. A significant correlation was observed between the number of IDPs in this experiment (Table II) and the LI (r = 0.63; P < 0.05) or NA (r = 0.74; P < 0.05) in the TEB structure of the mammary gland in experiment 1 (Table I). Discussion This study showed that the addition of high levels of Ca and Fe to a high-fat diet can increase, while PA alone or in combination with high levels of Ca and Fe can decrease the cell proliferative activity of both the mammary gland and the colon. The fact that the highest cell proliferative activity and more significant changes in the mammary gland were seen in the TEB than in the AB and TD structures is of significance since TEB is the structure which is the most sensitive to mammary carcinogens and often the site of tumor development (25—27). The NA in the mammary cells was similarly affected and it significantly related to the LI, indicating that the mammary cells undergoing DNA synthesis are of greater risk for nuclear damage by carcinogens. Although the pattern of changes in the number of IDPs was similar to those of the LI and NA, significant change after PA supplementation was seen only in the Ca-supplemented group, the group that exhibited the highest LI and NA in the mammary gland. The lack of significance in the IDP trends may in part be related to the time the animals were killed after DMBA exposure. In Sprague-Dawley rats, the IDP has been observed
33
*-B
7-9
10-12 13-13 18-18 1-3
»-«
7-9
1 0 - 1 2 1 3 - 1 5 IS-'.B
Fig. 1. Percent LI at various positions along the colonic crypt of mice fed various diets. For symbols see Table I.
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that in the TD or AB structures. There was a significant correlation between the LI and NA in the total structures of the mammary gland (r = 0.98; P < 0.01). No significant difference between the high-fat and low-fat groups was seen in the LI, MI and NA in the total and different structures of the mammary gland, with the exception of the LI in the TEB structure where the high-fat group had significantly higher values (Table I). As was seen in the mammary gland, the LI in the colon epithelial cells was significantly higher in the HCa and HFe groups compared with the high-fat group, and significantly lower in all PA supplemented diet groups compared with the high-fat, HCa and HFe groups (Table II). Examination of the labeled cell distribution in the colonic crypt (Figure 1) showed a shift in the labeled cells to higher crypt cell position when dietary fat level was increased. The addition of PA to the high-fat diet shifted the labeled cells to lower crypt cell positions, which are similar to those of the low-fat diet. The addition of Fe and particularly Ca to the high-fat diet shifted the proliferative zone to a higher level but this was reversed after the addition of PA. A significant correlation (r = 0.97; P < 0.01) was observed
L.U.Thompson and L.Zhang
The mechanisms whereby PA reduced the cell proliferation 2044
and IDP formation in the mammary epithelium is probably in part analogous to those discussed for the colon. Because PA is not readily absorbed or digested in the small intestine in the absence of dietary or intestinal phytases (58,59), its binding with Fe, Zn and Ca in the small intestine can decrease their absorption and availability to the mammary cell. The early markers of carcinogenesis did not differ, in most cases, between the low- and high-fat diet groups. This may be related to the fact that our feeding time was short, i.e. 2 weeks for cell proliferation and NA, and 3 weeks post-DMBA for IDP measurements, and the difference in fat level between the highand low-fat diets was small (5 and 25%). In previous studies, feeding times as long as 4 weeks for cell proliferation were used and the effect of 3 and 30% fat levels was compared (22). Furthermore, the total caloric intake and consequently the weight gain differed very little between the two groups. In this study, purified PA, which is soluble and known to be very reactive with minerals, was added. Whether the same effect can be seen when endogenous PA is used should be established in future experiments. Endogenous PA may exist as complexes with minerals or proteins, which remain insoluble under the gastrointestinal conditions; in such cases, PA may no longer be available for binding with additional minerals in the diet. In conclusion, we demonstrated, for the first time, an effect of PA, with low or high levels of minerals, on the mammary epithelial cells and the similarity in effect of PA on the proliferative activity of the colon and mammary epithelial cells in female mice. PA appears to reduce the risk for both mammary and colon carcinogenesis. Since its effect is more significant in the presence of large amounts of minerals, the mechanism may in part be related to its mineral-binding ability. PA may be one of the components of high-fiber diets responsible for their beneficial effect and its complete removal from food may need re-evaluation. Because a large concentration of PA has an adverse effect on nutrient availability (53,60), the PA and mineral concentrations in the diet that will decrease the cancer risk without compromising other health parameters should be determined in the future. Acknowledgements The authors thank Drs I.H.Russo and J.Russo for technical advice on the preparation of mammary tissue for histological analysis and the Natural Sciences and Engineering Research Council of Canada for financial assistance.
References 1. Drasar,B.S. and Irving,D. (1973) Environmental factors and cancer of the colon and breast. Br. J. Cancer, 11, 167—172. 2.Reddy,B.S., Cohen,L.A., McCoy.G.D., Hill.P., WeisburgerJ.H. and Wynder.E.L. (1980) Nutrition and its relationship to cancer. Cancer Res., 32, 237-245. 3. Howell.M.A. (1976) The association between colorectal cancer and breast cancer. J. Chron. Dis., 29, 243-261 4. Reddy.B.S. (1986) Diet and colon cancer: evidence for human and animal model studies. In Reddy.B.S. and Cohen,L.A. (eds), Diet, Nutrition and Cancer, Vol I: Macrorwlrieras and Cancer. CRC Press, Boca Raton, FL, pp. 47-66. 5. Cohen,L.A. (1986) Dietary fat and mammary cancer. In Reddy.B.S. and Cohen,L.A. (eds) Diet, Nutrition and Cancer, Vol. I: Macronutrients and Cancer. CRC Press, Boca Raton, FL, pp. 77-100. 6. Phillips.R.L. Garfinkel.K., KuzmanJ.W., Beeson.W.L., Tcrry,L. and Brin,B. (1980). Mortality among California Seventh Day Adventists for selected cancer sites. J. Nail Cancer Inst., 75, 1097-1107. 7. Graf,E. and Eaton.J.W. (1985) Dietary suppression of colonic cancer: fiber or phytate? Cancer, 36, 717-718. 8. Nielsen,B.N., Thompson,L.U. and Bird.R. (1987) Effect of phytic acid on colonic epithelial cell proliferation. Cancer Lett., 37, 317 — 325. 9. Shamsuddin,A.M., Hsayed,A.M. and Ullah.A. (1988) Suppression of large
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at 2 - 3 weeks post DMBA (25). In mice, IDP formation probably requires a longer time to develop than the 3 weeks used in this study. Nevertheless, since there were significant correlations between the number of IDP and LI as well as NA, all these parameters may be valuable early markers of risk for mammary carcinogens. The IDP results further indicate a possible cancer protective effect of PA in the mammary gland particularly in the presence of high levels of Ca. In case of the colon, not only reduced cell proliferation rate but also a shift in the proliferative compartment to a lower crypt position was seen in the presence of PA; this type of change has been suggested to be cancer protective (28-32). The enhancing effect of high dietary Fe levels on colonic cell proliferation and likely cancer risk seen in this study is in agreement with others (18,19,33) and may be related to the ability of Fe to catalyze lipid peroxidation and enhance the production of free radicals, which then can increase cellular damage (7,34-38). Several studies have shown that high Ca intake can reduce the cell proliferative activity and colon cancer incidence, and this has been attributed to the ability of Ca to bind with bile acids and fatty acids, suggested promoters of cell proliferation and tumor growth (39—44). Our results showed the contrary but find support in some recent works which showed increased colon tumor incidence and risk with high Ca supplementation (20-22, 45,46). The discrepancy may be related to (i) the level of Ca supplements used—many studies that showed protective effects used Ca levels < 1.0% (39-44), while our and other studies (20—22,45), which showed non-protective effects, used Ca levels of 1.0—1.6%; (ii) the type of calcium source used, i.e. CaHPO4 in our and other studies (20,45) versus calcium lactate, gluconate or carbonate in the other studies; or (iii) the type of fat used in the experimental protocol (22). Further research is needed to clarify the role of calcium supplements in carcinogenesis. In any case, excess calcium may enhance cell proliferation since it is involved in almost all cell cycle events (47—51). It may also influence lipid peroxidation (52). The suggested protective effect of PA in the colon seen in this study agrees with the results of other investigators (8-14). Nielsen et al. (8) showed that cell proliferation decreased as the level of the PA in the diet (0.6-2.0%) increased, while Shamsuddin and co-workers (9—13) showed that the addition of 1 or 2% PA in the drinking water of rats results in a significantly reduced tumor incidence and size compared with the controls. Several mechanisms have been suggested for this protective effect of PA in the colon (53), including (i) its ability to act as antioxidant by binding mineral catalysts of lipid peroxidatoin (7,18, 34,54); (ii) its binding with Zn, a cofactor of many enzymes involved in DNA synthesis (8,55); (iii) its binding with amylase enzymes or with starch, thereby increasing the amount of starch that is undigested and is fermented in the colon to short-chain fatty acids (56,57); this in turn can lower the pH to a cancer protective level; (iv) its ability to enhance the baseline natural killer cell activity (12); and (v) mechanisms involving the inositol triphosphate which is produced upon PA hydrolysis (10—13). Nevertheless, in this study, because PA reduced the cell proliferative activity in the colon of rats fed a high-fat diet, particularly when the levels of Fe and Ca were high, our data provide further evidence that the effect of PA in the colon is somehow linked to its mineral-binding ability. The reduced colon tumor incidence observed in rats fed PA in the presence of excess magnesium (14) or iron (19) agreed with our observations.
Ptiytic add and minerals tion of hyaluronic acid in the presence of ferritin. FEBS Lett., 177, 2 7 - 3 0 . 37. Halliwell,B. and Gutteridge,M.C. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219, 1-14. 38. Ames.B.N. (1983) Dietary carcinogens and anticarcirtogens. Science, 221, 1256-1264. 39.Sorcnson,A.W., Slattery.M.L. and Ford.M.H. (1988) Calcium and colon cancer: a review. Nutr. Cancer, 11, 135-145. 40. Wargovich.M.J. (1988) Calcium and colon cancer. J. Am. Coll. Nutr., 7, 295-300. 41. Reshef.R., Rozen.P., Fireman,Z., Fine,W., Barzilai.M., Shasha.S.M. and Shkolnik.T. (1990) Effect of calcium enriched diet on the colonic epithelium hyperproliferation induced by /V-methyl A^-nitrosoguanidine in rats in a low calcium and fat diet. Cancer Res., 50, 1764-1767. 42 Wargovich.M.J., AUnutt.D., Palmer.C, Anaya.P. and Stephens.L.C. (1990) Inhibition of the promotional phase of azoxymethane induced colon carcinogenesis in the F344 rat by calcium lactate: effect of simulating two human nutrient density levels. Cancer Lett., 53, 17-25. 43. App!eton,G.V.N., Davies.P.W., BristoU.B. and Williamson.R.C.N. (1987) Inhibition of intestinal carcinogenesis by dietary supplementation with calcium. Br. J. Surg., 74, 523-535. 44. Pence,B.C. and Buddingh.F. (1988) Inhibition of dietary fat promoted colon carcinogenesis in rats by supplemental calcium or vitamin D3. Carcinogenesis, 91, 187-190. 45. Bull.A., Bird.R.P., Bruce,W.R., Nigro.N. and Medline.A. (1987) Effect of calcium on azoxymethane induced tumors in rats. Gastroenterology, 92, 1332. 46. Gregoire.R.C, Stern,H.S., Yeung.K.S., StadlerJ., Langlcy.S. Furrer.R. and Bruce,W.R. (1989) Effect of calcium supplementation on mucosal cell proliferation in high risk patients for colon cancer. Gut, 30, 376-382. 47. Leffert,H.L. (1982) Monovalent cations, cell proliferation and cancer an overview. In Boynton,W.L., McKeehan.K.A. and WhitfieldJ.F. (eds), Anions, Cell Proliferation and Cancer. Academic Press, New York, pp. 93-102. 48. Whitfield,J.F. (1982) The role of calcium and magnesium in cell proliferation: an overview. In Boynton.W.L., McKeehan.K.A. and Whitfield.J.F. (eds), Anions, Cell Proliferation and Cancer. Academic Press, New York, pp. 283-294. 49. Norris.V. (1989) A calcium flux at the termination of replication triggers cell division in Escherichia coli. Cell Calcium, 10, 511-517. 50. CameronJ.L. and Smith,N.K.R. (1989) Cellular concentration of magnesium and other ions in relation to protein synthesis, cell proliferation and cancer. Magnesium, 8, 31—44. 51. Tucker.R.W., Meade-Cobun,K. and Ferris.D. (1990) Cell shape and increased free cytosolic calcium [Ca+2]i induced by growth factors. Cell Calcium, 11, 201-209. 52. Braughler,J.M. (1987) Calcium and lipid peroxidation. Upjohn Symposium/Oxygen Radicals. The Upjohn Co. Kalamazoo MI, pp. 99—104. 53. Thompson,L.U. (1989) Nutritional and physiological effects of phytic acid. In KinsellaJ.E. and Soucie.W. (eds), Food Proteins, AOCS, Champaign.IL, pp. 410-431. 54. Thompson,H.J., Kennedy.K., Witt,M. and Jusefyk J. (1991) Effect of dietary iron deficiency or excess on the induction of mammary carcinogenesis by 1-methyl-l-nitrosourea. Carcinogenesis, 12, 111-114. 55. Southon.S., Livesey.G., GeeJ. and Johnson.I.T. (1985) Intestine cellular proliferation and protein synthesis in Zn-deficient rats. Br. J. Nutr., 53, 595-602. 56. Thompson,L.U. (1988) Antinutrients and blood glucose. Food Techno!., 42, 123-132. 57. Thompson,L.U., Button.C.L. and Jenkins,D.J.A. (1987) Phytic acid and calcium affects starch digestibility and blood glucose response in humans. Am. J. Clin. Nutr., 46, 467-473. 58. SandbergAS. and Andersson.H. (1988) Effect of dietary phytase on the digestion of phytate in the stomach and small intestine of humans. J. Nutr., 118, 469-473. 59. Anonymous (1989) Phytase and phytate degradation in humans. Nutr. Rev., 47, 155-157. 60. Spivey-Fox,M.R. and Tao.S.H. (1989) Antinutritive effects of phytate and other phosphorylated derivatives. In HancockJ. (ed.), Nutritional Toxicology. Academic Press, New York, Vol. 3, pp. 59-96. Received on March 4, 1991; revised on August 16, 1991; accepted on August 21, 1991
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intestinal cancer in F-344 rats by inositol hexaphosphate. Carcinogenesis, 9, 577-580. 10. Shamsuddin.A.M. and Ullah.A. (1989) Inositol hexaphosphate inhibits large intestinal cancer in F-344 rats 5 months following induction by azoxymethane. Carcinogenesis, 10, 625—626. 11. Shamsuddin.A.M., Ullah.A. and Chakvarthy.A. (1989) Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation CD-I mice. Carcinogenesis, 10, 1461-1463. 12. Baten.A., Ullah.A., Tomazic.V.J. and Shamsuddin.A.M. (1989) Inositol phosphate induced enhancement of natural killer cell activity correlates with tumor suppressions. Carcinogenesis, 10, 1595-1598. 13. Ullah,A. and Shamsuddin.A.M. (1990) Dose-dependent inhibition of large intestinal cancer by inositol hexaphosphate in F344 rats. Carcinogenesis, 11, 2219-2222. 14. Jariwalla.R.J., Sabin.R., Lawson.S., Bloch,D.A., Prender,M., Andrews.V. and Herman,Z.S. (1988) Effect of dietary phytic acid on the incidence and growth rate of tumors promoted in Fisher rats by Mg supplement. Nutr. Res., 8, 813-827. 15. American Institute of Nutrition (1977) Report of the Ad Hoc Committee on standards of nutritional studies. J. Nutr., 107, 1340—1348. 16. Bindra.G., Gibson,R. and Thompson,L.U. (1986) [Phytate][Ca/Zn] molar ratios in Asian immigrant lacto-ovo vegetarian diets and their relationship to Zn nutnture. Nutr. Res., 6, 475-483. 17. Bird,R.P. and Stamp.D. (1986) Effect of a high fat diet on the proliferative indices of murine colonic epithelium. Cancer Lett., 31, 61—67. 18. Siegers.C.P., Buman,D., Baretton.G. and Younes.M. (1988) Dietary iron enhances tumor rate in dimethylhydrazine-induced colon carcinogenesis in mice. Cancer Lett., 41, 251-256. 19. Nelson,R.L., Yoo.S.J., TanureJ.C, Andrianopoulos,G. and Misumi,A. (1989) The effect iron on experimental colorectal carcinogenesis. AnriCancer Res., 9, 1477-1482 20 McSherry.C.S., Cohen.B.I., Bokkenheuser.Y.D., Mosbach,E.H., WinterJ., Matoba,N. and ScholesJ. (1989) Effect of calcium, and bile acid feeding on colon tumors in the rat. Cancer Res., 49, 5039-6043 21. Kaup.S.M., Behling.A.R., Choquette,L.L. and Greger.J.L. (1989) Colon tumor development in DMH-initiated rats fed varying levels of calcium and butterfat. FASEB J., 3, A469. 22. Behling,A.R., Kaup,S., Choquette.L.L. and GregorJ.L. (1990) Lipid absorption and intestinal tumor incidence in rats fed varying levels of calcium and butterfat. Br. J. Nutr., 64, 505-513. 23. Zhang,L., Bird.R.P. and Bruce,W.R. (1987) Proliferative activity of munne mammary epithelium as affected by dietary fat and calcium. Cancer Res., 47, 4905-4908. 24. Sharkey.S.M. and Bruce.W.R. (1986) Quantitation of nuclear aberration as a screen for agents damaging to mammary epithelium. Carcinogenesis, 7, 1991-1995. 25. Russo,I.H. and Russo,J (1978) Developmental stage of rat mammary gland as determinant of its susceptibility to 7,12 dimethylbenzanthracene. J. Nail. Cancer Inst., 61, 1439-1449. 26. Russo.I H. and RussoJ. (1987) Biology of disease: biological and molecular basis of mammary carcinogenesis. Lab. Invest., 57, 112-137. 27. RussoJ.H., Gusterson,A., Rogers,A.E, Russo.I.H., Wellings,S.R. and van Switen.M.J. (1990) Biology of disease: comparative study of human and rat mammary tumorigenesis. Lab. Invest., 62, 244—278. 28. Lipkin.M., Vehara,K., Winawar,S., Sanchez,A., Bauer.C, Lynch,H., Blattner,W. and Faumeni J. (1985) Seventh Day Adventist vegetarians have quiescent proliferative activity in colonic mucosa. Cancer Lett., 26, 139-144. 29. Lipkin.M (1987) Biomarkers of increased susceptibility to gastrointestinal cancer. The development and application to studies of cancer prevention. Gastroenterology, 92, 1083-1086. 30. Romagnoli.P., Filippino,F., Bandettini,L. and Brugnola,D. (1984) Increase of mitotic activity in the colonic mucosa of patients with colorectal cancer. Dis. Colon Rectum, 27, 305-308. 31. Deschner.E.E. and Maskens.A.P. (1982) Significance of the labelling index and labelling index distribution as kinetic parameters in colorectal mucosa of cancer patients and DMH treated animals. Cancer, 50, 1136- 1141. 32.Sugarbaker,P.H., MacdonaldJ.S. and Gunderson.L.L. (1982) Colorectal cancer. In DeVita.V.T., Jr, Hellman.S. and Rosenberg,S.A. (eds), Cancer: Principles and Practices of Oncology. Lippincott, Toronto, pp. 643—676. 33. Stevens.R.G., Jones,D.Y., Mocozzi.M.S. andTaylor.P.R. (1988) Body iron stores and the risk of cancer. New Engl. J. Med., 319, 1047-1052. 34. Graf.E., MahoneyJ.R., Bryant.R.G. and Eaton J.W. (1984) Iron catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site. J. Biol Chem., 259, 3620-3624. 35. Bannister J.V., Barmister.W.H. and Thomally.PJ. (1984) The effect of ferritin iron loading on hydroxyl radical production. Life Chem Rep., 2, 64-72. 36. Carlin,G. and Djursater,R. (1984) Xanthine oxidase induced depolymerisa-
Phytic acid and minerals: effect on early markers of risk for mammary and colon carcinogenesis
Lilian U.Thompson and Lin Zhang Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
Introduction Epidemiological studies have shown a close relationship between colon and breast cancers (1-3) and a possibility that they have some common etiologic factors has been suggested. Certain dietary factors such as fat have been implicated as enhancing agents in both cancers (1,4,5). However, the fact that dietary fiber intake is high in vegetarians or semi-vegetarians who have lower cancer incidence than omnivores or non-vegetarians (1,2,6) indicates that dietary fiber or some substances associated with it such as inositol hexaphosphate or phytic acid (PA) may have some cancer protective effect (1,2,6,7). Previous work in our laboratory and others have shown that PA can lower the cell proliferative activity and tumor incidence and growth in the colon of rats and mice (8 -14). It is not known whether PA can also affect mammary cancer. However, because of the close relationship between the two cancers, we hypothesize 'Abbreviations: PA, inositol hexaphosphate or phytic acid; NA, nuclear aberration; IDP, intraductal proliferation; DMBA, dimethylbenzanthracene; LI, labeling index; MI, mitotic index; TEB, terminal end bud; AB, alveolar bud; TD, terminal duct. Diets: H, high-fat diet; HPA, diet H with PA; HCa, diet H with high Ca level; HFe, diet H with high Fe level; HPACA, diet H with PA and high Ca level; HPAFe, diet H with PA and high Fe level.
Materials and methods Animals In two experiments, 3-week old female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were used. The animals were housed in wire-top plastic cages maintained at 23°C with 12 h light/dark cycle. Diets and deionized water were provided ad libitum. Experimental diets All diets were based on the AIN-76 composition (15) wrth modifications for type of carbohydrate (only com starch instead of sucrose and com starch) and level of fat, minerals and PA used. Experimental diet L, was a low-fat control diet containing 5% com oil, while diet H was a high-fat control diet with 25% corn oil. Diets HCa, HFe, HPA, HPACa, and HPAFe were all high-fat (25%) diets with 1.5% total Ca (as CaHPO4), 535 p.p.m. total Fe (as FeSO4-7 H2O), 1.2% PA (as sodium phytate) alone or PA in combination with the Ca or Fe respectively. Fat, PA, Fe and Ca were added at the expense of corn starch. All diet ingredients were obtained from ICN Biochemicals (Costa Mesa, CA) except com starch (St Lawrence Starch Ltd, Mississauga, Ontario), com oil (Mazola, Toronto, Ontario), sodium phytate (Sigma Chemical Co, St Louis, MO), CaHPO4 and FeSO-7 H2O (Fisher Scientific Co., Toronto, Ontario). The control diets contained 0.5% Ca, 35 p.p.m. Fe and no PA; therefore, the experimental design essentially tested the effect of two levels (i.e. high and low) of the additives. The 1.2% PA level may be approached in a lacto-ovo-vegetarian diet (8,16). The high PA, Ca and Fe levels were chosen because they are close to the levels that have been shown individually to cause some effect on the colon or mammary ceils (8-14, 17-22) and, hence, are ideal in demonstrating interactions. A high-fat diet was used as the primary control since it is known to increase cancer risk (1,4,5). A low-fat diet was included as an additional control since it has been associated with lower risk for carcinogenesis. Experiment 1 Eighty-four female mice were randomized into seven groups, acclimatized for 1 week and then fed their special diets and deionized water for 2 weeks. Body weights were monitored during the feeding period. At the end of the feeding period, half of the animals per group were given injections of ccJchicine (1 /jg/g body wt; Sigma) and [3H]thymidine (2 fiCi/g, body wt; Amersham Corp. Arlington Heights, IL) and 2 h later were killed. The thoracic mammary glands and colon tissues were removed, stretched flat on a filter paper, fixed in 10% neutral formalin and prepared for histcJogical examination. Mammary gland and colon tissues were embedded in paraffin blocks, sectioned ( 4 - 5 jim) and transferred to slides. One set of slides was dewaxed, processed for autoradiography using standard techniques (8,17,23) and examined microscopically for number and position of labeled cells. Another set of slides was dewaxed, directly stained with H&E and examined for number of cells undergoing mitosis. The other half of the animals per group were fasted overnight and given orally a single dose of the mammary carcinogen dimethylbenzanthracene (DMBA; 100
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This study determined the effect of inositol hexaphosphate or phytic acid (PA; 1.2%), Ca (1.5%) and Fe (535 p.p.m.) alone, and PA in combination with Ca or Fe in a high-fat diet (25%) on the labeling (LI) and mitotk (MI) cell proliferation indices, nuclear aberration (NA) and intraductal proliferation (IDP) in the mammary gland, as well as the LI in colonic epithelial cells. Diet supplementation with PA alone caused reductions (P < 0.05) in the colon LI by 18%, and in the LI and NA in the total mammary gland structures of mice by 29 and 30% respectively. Supplementation with Fe or particularly Ca caused increases hi the colon LI and in the mammary LI, MI, NA and IDP but these were reduced by 25-53% (P < 0.05) in the presence of PA. These results support the hypothesis that PA may reduce the risk for both colon and mammary cancer and its effect is related to its mineral binding ability. Furthermore, significant relationships (P < 0.01) were observed between the LI and MI or NA in the total structures of the mammary gland. The number of IDPs also related (P < 0.05) to LI or NA in the terminal end bud structure of the mammary gland, suggesting that highly proliferating mammary cells, particularly in the terminal end bud structure, are of greater risk for nuclear damage and development to IDP. A significant relationship (P < 0.01) was observed between the cell proliferation in the mammary gland and that in the colon, indicating that both tissues can be influenced similarly by dietary constituents.
that a similar protective effect for mammary cancer may also be observed. The mechanisms whereby PA may affect cancer development is not clear. Nevertheless, since PA is a highly negatively charged molecule that can bind with minerals such as Fe and Ca, we also hypothesize that its effect may be partly related to its mineral binding ability. Therefore the overall objective of this study was to determine the effect of PA on early markers of risk for mammary and colon carcinogenesis, and to determine if the effect is related to the PA's mineral binding ability. Specifically, we determined the effect of PA, Ca and Fe alone, or PA in combination with Ca or Fe on (i) the cell proliferation, nuclear aberration (NA) and intraductal proliferation (IDP) in the mammary gland and (ii) cell proliferation in the colonic epithelial cell. We also correlated the cell proliferative activities in the colon and in the mammary gland.
L.U.Thompson and L.Zhang
Experiment 2 Fifty-six female mice were divided into seven diet groups, acclimatized for 1 week and fed their special diets for 2 weeks as in experiment 1. After fasting overnight, they were given orally DMBA (100 mg/kg body wt) in com oil, and then given back their same special diet for another 3 weeks. Body weight was monitored three times a week during the feeding period. At the end of the experiment, mammary glands were removed, sectioned, stained with H&E as above, and microscopically examined for IDP (25-27). When DMBA is introduced when TEBs are plentiful and actively differentiating into ABs, many TEBs do not eventually differentiate; instead they become larger and at this stage are called the IDPs (25-27). While TEBs are characterized by 3 - 6 layers of epithelial cell lining or layer, the IDP has 6—10 layers. Confluence of IDP leads to the formation of a microtumor, which in time becomes a palpable tumor. Statistical analysis The mean standard error of the mean were calculated for each diet groups. Statistical differences between groups in LI, MI, NA and IDP formation in the breast and/or colon tissues were calculated by one-way analysis of variance followed by Duncan's multiple range test. Regression analysis was done between the risk marker indices.
Table I. Effect of various diets on LJ, MI and NA in the total and different structures of the mammary gland Diet group*
Terminal end bud
L H HFe HCa HPA HPAFe HPACa
14.41 17.22 20.11 20.04 11.59 11.66 12.50
± ± ± ± ± ± ±
0.73 c 0.80 b 0.42" 0.63" 0.6^ 0.67 d 0.81 d
2.73 3.08 4.03 3.64 2.00 1.51 1.77
± ± ± ± ± ± ±
L H HFe HCa HPA HPAFe HPACa
3.17 3.51 4.71 5.19 2.80 2.95 3.19
± ± ± ± ± ± ±
0.22 b 0.47 b 0.41" 0.51' 0.11 b 0.22 b 0.10 b
0.98 1.04 1.61 2.05 0.91 0.81 0.93
± ± ± ± ± ± ±
L H HFe HCa HPA HPAFe HPACa
7.06 8.30 9.16 9.53 6.45 6.81 6.86
± ± ± ± ± ± ±
0.20 b 0.86 < b 0.64 1 0.77* 0.29 b 0.71 b 0.34 b
0.93 0.76 0.86 0.90 0.65 0.69 0.79
± ± ± ± ± ± ±
Terminal duct LI 0.61 b ' c d 0.28*'b'c 0.29* 0.56 l b
o.ird d 0.33 0.29"1
Alveolar bud
Total
1.77 1.75 2.51 2.12 1.20 1.36 0.89
± 0.38lbc ± 0.22"'b-c ± 0.45" ± 0.41" b ± 0.1 l c ±O.32 b - c ± 019bC
7.15 8.06 9.36 9.46 5.69 5.58 5.52
± ± ± ±
0.20"
0.25 0.43 0.47 0.95 0.28 0.28 0.35
± ± ± ± ± ± ±
0.07 b 0.15 b 0.14 b 0.09* 0.08 b 0.03 b 0.05 b
2.19 2.36 2.87 3.74 1.53 1.81 1.77
± ± ± ± ± ±
0.16bc 0.33bc 0.29 b 0.50" 0.09 c 0.13°
*
o.or
2.43 2.19 2.04 2.56 1.56 1.81 1.92
± 0.15lb ± 0.24 l b 0.26 b c ± 0.11" ± 0.18° ± 0.2 l b c ± 0.11 b c
3.72 4.56 4.71 4.87 3.19 3.36 3.11
± 0.13 b c 0.34 l b ± 0.41* b ± 0.42" ± 0.18° ± 0.46c ± 0.19°
O.SO1' 0.32" 0.45" O.33 c ± 0.32 c ± 0.44 c
MI 0.13 b 0.09" O.35 l b 0.50" 0.09 b 0.18 b 0.06" NA 0.07* 0.1 l " b 0.07*-b 0.1 l " b 0.04b 0.06 l b 0.03lb
•L, low fat (5%); H, high fat (25%); HPA, high fat with 1.2% PA; HFe, high fat with 535 p.p.m. Fe; HCa, high fat with 1.5% Ca; HPAFe, high fat with 1.2% PA and 535 p.p.m. Fe; HPACa, high fat with 1.2% PA and 1.5% Ca. Means within the same column with different superscripts are significantly different (P < 0.05).
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Results Experiment I No significant difference in the final body weight was observed between diet groups. While all high-fat diet groups also did not differ significantly with each other in weight gain, they had - 14% higher values (P < 0.05) than the low-fat diet group. Table I shows that high Ca or Fe supplementation of the highfat diet significantly increased the LI in the total structures of the mammary gland, while PA supplementation alone or in combination with Ca or Fe significantly decreased it. When the location of the LJ in the different mammary gland structures was examined, a similar pattern of changes were observed. However, not only was the LI value the highest in the TEB, but also more significant differences in LJ were seen in the TEB compared with in the TD and the AB structures, e.g. while the differences in LJ between the H and HFe, HCa, HPA, HPAFe or HPACa, between HFe and HPAFe and between HCa and HPACa groups were significant in the TEB, only the last four differences were significant in the TD and only the difference between HFe and HPAFe groups was significant in the AB structure. Regarding the MI, a similar trend as the LJ was seen in both the total and in the different structures of the mammary gland (Table I); thus, a significant relationship (r = 0.92; P < 0.01) was observed between the LI and MI in the total structures of the mammary gland. The NA was significantly higher in the total mammary gland structures of the high-fat and mineral-supplemented diet groups compared with all the PA-supplemented diet groups (Table I). In all groups, the NA was the highest in the TEB compared to
mg/kg body wt) in 0.2 ml com oil as established by Sharkey and Bruce (24). After 24 h, the animals were killed and their mammary glands were removed, fixed in formalin, prepared for histology, and directly stained with H&E for examination of NA as previously described (24). The labeling index (LI), indicating the number of epithelial cells undergoing DNA synthesis, was estimated as the percentage of epithelial cells labeled by tritiated thymidine, while the mhotic index (MI) was estimated as the percentage of cells undergoing mitosis. NA is the percentage of epithelial cells with nuclear aberration which can be seen in the mammary tissues after DMBA treatment. The LI, MI and NA in the total and different structures of the mammary gland, i.e. terminal end bud (TEB), alveolar bud (AB) and terminal duct (TD), were determined. The nature and appearance of these structures have been well described (25-27). The AB is formed from the normal differentiation of the TEB structure. A large number of the TEBs do not differentiate, become progressively smaller and form the finger-shaped TD structures.
Ptiytk add and minerals
Table n. Effect of various diets on the LI in the colon epithelial cells and the IDP in the mammary gland of mice Diet groups'
Colon LI (experiment 1)
L H HFe HCa HPA HPAFe HPACa
10.15 10.98 13.92 13.49 9.03 9.11 9.22
± ± ± ± ± ± ±
IDP (experiment 2)
0.28 bc 0 31 b O.571 0.32* 0.10° 0.39* 0.34c
1.50 2.88 4.50 5.38 3.00 3.38 3.14
± ± ± ± ± ± ±
0.06C 0.35 b c 0.87* b 0.73* 0.53 b c 0.68 b c 0.52 b c
•For symbols, see Table I. Means within the same column with different superscripts are significantly different (P < 0.05)
I
between the LI in the total structure of the mammary gland (Table I) and the LI in the colon epithelial cells (Table II). Experiment 2 No significant differences in final body weight and weight gain were seen between diet groups. The high-fat group did not differ in number of IDPs compared with all the other groups, with the exception of the HCa group which had a significantly higher number of IDPs (Table H). PA supplementation, however, significantly decreased the number of IDPs in the group fed the high Ca-supplemented diet. A significant correlation was observed between the number of IDPs in this experiment (Table II) and the LI (r = 0.63; P < 0.05) or NA (r = 0.74; P < 0.05) in the TEB structure of the mammary gland in experiment 1 (Table I). Discussion This study showed that the addition of high levels of Ca and Fe to a high-fat diet can increase, while PA alone or in combination with high levels of Ca and Fe can decrease the cell proliferative activity of both the mammary gland and the colon. The fact that the highest cell proliferative activity and more significant changes in the mammary gland were seen in the TEB than in the AB and TD structures is of significance since TEB is the structure which is the most sensitive to mammary carcinogens and often the site of tumor development (25—27). The NA in the mammary cells was similarly affected and it significantly related to the LI, indicating that the mammary cells undergoing DNA synthesis are of greater risk for nuclear damage by carcinogens. Although the pattern of changes in the number of IDPs was similar to those of the LI and NA, significant change after PA supplementation was seen only in the Ca-supplemented group, the group that exhibited the highest LI and NA in the mammary gland. The lack of significance in the IDP trends may in part be related to the time the animals were killed after DMBA exposure. In Sprague-Dawley rats, the IDP has been observed
33
*-B
7-9
10-12 13-13 18-18 1-3
»-«
7-9
1 0 - 1 2 1 3 - 1 5 IS-'.B
Fig. 1. Percent LI at various positions along the colonic crypt of mice fed various diets. For symbols see Table I.
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that in the TD or AB structures. There was a significant correlation between the LI and NA in the total structures of the mammary gland (r = 0.98; P < 0.01). No significant difference between the high-fat and low-fat groups was seen in the LI, MI and NA in the total and different structures of the mammary gland, with the exception of the LI in the TEB structure where the high-fat group had significantly higher values (Table I). As was seen in the mammary gland, the LI in the colon epithelial cells was significantly higher in the HCa and HFe groups compared with the high-fat group, and significantly lower in all PA supplemented diet groups compared with the high-fat, HCa and HFe groups (Table II). Examination of the labeled cell distribution in the colonic crypt (Figure 1) showed a shift in the labeled cells to higher crypt cell position when dietary fat level was increased. The addition of PA to the high-fat diet shifted the labeled cells to lower crypt cell positions, which are similar to those of the low-fat diet. The addition of Fe and particularly Ca to the high-fat diet shifted the proliferative zone to a higher level but this was reversed after the addition of PA. A significant correlation (r = 0.97; P < 0.01) was observed
L.U.Thompson and L.Zhang
The mechanisms whereby PA reduced the cell proliferation 2044
and IDP formation in the mammary epithelium is probably in part analogous to those discussed for the colon. Because PA is not readily absorbed or digested in the small intestine in the absence of dietary or intestinal phytases (58,59), its binding with Fe, Zn and Ca in the small intestine can decrease their absorption and availability to the mammary cell. The early markers of carcinogenesis did not differ, in most cases, between the low- and high-fat diet groups. This may be related to the fact that our feeding time was short, i.e. 2 weeks for cell proliferation and NA, and 3 weeks post-DMBA for IDP measurements, and the difference in fat level between the highand low-fat diets was small (5 and 25%). In previous studies, feeding times as long as 4 weeks for cell proliferation were used and the effect of 3 and 30% fat levels was compared (22). Furthermore, the total caloric intake and consequently the weight gain differed very little between the two groups. In this study, purified PA, which is soluble and known to be very reactive with minerals, was added. Whether the same effect can be seen when endogenous PA is used should be established in future experiments. Endogenous PA may exist as complexes with minerals or proteins, which remain insoluble under the gastrointestinal conditions; in such cases, PA may no longer be available for binding with additional minerals in the diet. In conclusion, we demonstrated, for the first time, an effect of PA, with low or high levels of minerals, on the mammary epithelial cells and the similarity in effect of PA on the proliferative activity of the colon and mammary epithelial cells in female mice. PA appears to reduce the risk for both mammary and colon carcinogenesis. Since its effect is more significant in the presence of large amounts of minerals, the mechanism may in part be related to its mineral-binding ability. PA may be one of the components of high-fiber diets responsible for their beneficial effect and its complete removal from food may need re-evaluation. Because a large concentration of PA has an adverse effect on nutrient availability (53,60), the PA and mineral concentrations in the diet that will decrease the cancer risk without compromising other health parameters should be determined in the future. Acknowledgements The authors thank Drs I.H.Russo and J.Russo for technical advice on the preparation of mammary tissue for histological analysis and the Natural Sciences and Engineering Research Council of Canada for financial assistance.
References 1. Drasar,B.S. and Irving,D. (1973) Environmental factors and cancer of the colon and breast. Br. J. Cancer, 11, 167—172. 2.Reddy,B.S., Cohen,L.A., McCoy.G.D., Hill.P., WeisburgerJ.H. and Wynder.E.L. (1980) Nutrition and its relationship to cancer. Cancer Res., 32, 237-245. 3. Howell.M.A. (1976) The association between colorectal cancer and breast cancer. J. Chron. Dis., 29, 243-261 4. Reddy.B.S. (1986) Diet and colon cancer: evidence for human and animal model studies. In Reddy.B.S. and Cohen,L.A. (eds), Diet, Nutrition and Cancer, Vol I: Macrorwlrieras and Cancer. CRC Press, Boca Raton, FL, pp. 47-66. 5. Cohen,L.A. (1986) Dietary fat and mammary cancer. In Reddy.B.S. and Cohen,L.A. (eds) Diet, Nutrition and Cancer, Vol. I: Macronutrients and Cancer. CRC Press, Boca Raton, FL, pp. 77-100. 6. Phillips.R.L. Garfinkel.K., KuzmanJ.W., Beeson.W.L., Tcrry,L. and Brin,B. (1980). Mortality among California Seventh Day Adventists for selected cancer sites. J. Nail Cancer Inst., 75, 1097-1107. 7. Graf,E. and Eaton.J.W. (1985) Dietary suppression of colonic cancer: fiber or phytate? Cancer, 36, 717-718. 8. Nielsen,B.N., Thompson,L.U. and Bird.R. (1987) Effect of phytic acid on colonic epithelial cell proliferation. Cancer Lett., 37, 317 — 325. 9. Shamsuddin,A.M., Hsayed,A.M. and Ullah.A. (1988) Suppression of large
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at 2 - 3 weeks post DMBA (25). In mice, IDP formation probably requires a longer time to develop than the 3 weeks used in this study. Nevertheless, since there were significant correlations between the number of IDP and LI as well as NA, all these parameters may be valuable early markers of risk for mammary carcinogens. The IDP results further indicate a possible cancer protective effect of PA in the mammary gland particularly in the presence of high levels of Ca. In case of the colon, not only reduced cell proliferation rate but also a shift in the proliferative compartment to a lower crypt position was seen in the presence of PA; this type of change has been suggested to be cancer protective (28-32). The enhancing effect of high dietary Fe levels on colonic cell proliferation and likely cancer risk seen in this study is in agreement with others (18,19,33) and may be related to the ability of Fe to catalyze lipid peroxidation and enhance the production of free radicals, which then can increase cellular damage (7,34-38). Several studies have shown that high Ca intake can reduce the cell proliferative activity and colon cancer incidence, and this has been attributed to the ability of Ca to bind with bile acids and fatty acids, suggested promoters of cell proliferation and tumor growth (39—44). Our results showed the contrary but find support in some recent works which showed increased colon tumor incidence and risk with high Ca supplementation (20-22, 45,46). The discrepancy may be related to (i) the level of Ca supplements used—many studies that showed protective effects used Ca levels < 1.0% (39-44), while our and other studies (20—22,45), which showed non-protective effects, used Ca levels of 1.0—1.6%; (ii) the type of calcium source used, i.e. CaHPO4 in our and other studies (20,45) versus calcium lactate, gluconate or carbonate in the other studies; or (iii) the type of fat used in the experimental protocol (22). Further research is needed to clarify the role of calcium supplements in carcinogenesis. In any case, excess calcium may enhance cell proliferation since it is involved in almost all cell cycle events (47—51). It may also influence lipid peroxidation (52). The suggested protective effect of PA in the colon seen in this study agrees with the results of other investigators (8-14). Nielsen et al. (8) showed that cell proliferation decreased as the level of the PA in the diet (0.6-2.0%) increased, while Shamsuddin and co-workers (9—13) showed that the addition of 1 or 2% PA in the drinking water of rats results in a significantly reduced tumor incidence and size compared with the controls. Several mechanisms have been suggested for this protective effect of PA in the colon (53), including (i) its ability to act as antioxidant by binding mineral catalysts of lipid peroxidatoin (7,18, 34,54); (ii) its binding with Zn, a cofactor of many enzymes involved in DNA synthesis (8,55); (iii) its binding with amylase enzymes or with starch, thereby increasing the amount of starch that is undigested and is fermented in the colon to short-chain fatty acids (56,57); this in turn can lower the pH to a cancer protective level; (iv) its ability to enhance the baseline natural killer cell activity (12); and (v) mechanisms involving the inositol triphosphate which is produced upon PA hydrolysis (10—13). Nevertheless, in this study, because PA reduced the cell proliferative activity in the colon of rats fed a high-fat diet, particularly when the levels of Fe and Ca were high, our data provide further evidence that the effect of PA in the colon is somehow linked to its mineral-binding ability. The reduced colon tumor incidence observed in rats fed PA in the presence of excess magnesium (14) or iron (19) agreed with our observations.
Ptiytic add and minerals tion of hyaluronic acid in the presence of ferritin. FEBS Lett., 177, 2 7 - 3 0 . 37. Halliwell,B. and Gutteridge,M.C. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219, 1-14. 38. Ames.B.N. (1983) Dietary carcinogens and anticarcirtogens. Science, 221, 1256-1264. 39.Sorcnson,A.W., Slattery.M.L. and Ford.M.H. (1988) Calcium and colon cancer: a review. Nutr. Cancer, 11, 135-145. 40. Wargovich.M.J. (1988) Calcium and colon cancer. J. Am. Coll. Nutr., 7, 295-300. 41. Reshef.R., Rozen.P., Fireman,Z., Fine,W., Barzilai.M., Shasha.S.M. and Shkolnik.T. (1990) Effect of calcium enriched diet on the colonic epithelium hyperproliferation induced by /V-methyl A^-nitrosoguanidine in rats in a low calcium and fat diet. Cancer Res., 50, 1764-1767. 42 Wargovich.M.J., AUnutt.D., Palmer.C, Anaya.P. and Stephens.L.C. (1990) Inhibition of the promotional phase of azoxymethane induced colon carcinogenesis in the F344 rat by calcium lactate: effect of simulating two human nutrient density levels. Cancer Lett., 53, 17-25. 43. App!eton,G.V.N., Davies.P.W., BristoU.B. and Williamson.R.C.N. (1987) Inhibition of intestinal carcinogenesis by dietary supplementation with calcium. Br. J. Surg., 74, 523-535. 44. Pence,B.C. and Buddingh.F. (1988) Inhibition of dietary fat promoted colon carcinogenesis in rats by supplemental calcium or vitamin D3. Carcinogenesis, 91, 187-190. 45. Bull.A., Bird.R.P., Bruce,W.R., Nigro.N. and Medline.A. (1987) Effect of calcium on azoxymethane induced tumors in rats. Gastroenterology, 92, 1332. 46. Gregoire.R.C, Stern,H.S., Yeung.K.S., StadlerJ., Langlcy.S. Furrer.R. and Bruce,W.R. (1989) Effect of calcium supplementation on mucosal cell proliferation in high risk patients for colon cancer. Gut, 30, 376-382. 47. Leffert,H.L. (1982) Monovalent cations, cell proliferation and cancer an overview. In Boynton,W.L., McKeehan.K.A. and WhitfieldJ.F. (eds), Anions, Cell Proliferation and Cancer. Academic Press, New York, pp. 93-102. 48. Whitfield,J.F. (1982) The role of calcium and magnesium in cell proliferation: an overview. In Boynton.W.L., McKeehan.K.A. and Whitfield.J.F. (eds), Anions, Cell Proliferation and Cancer. Academic Press, New York, pp. 283-294. 49. Norris.V. (1989) A calcium flux at the termination of replication triggers cell division in Escherichia coli. Cell Calcium, 10, 511-517. 50. CameronJ.L. and Smith,N.K.R. (1989) Cellular concentration of magnesium and other ions in relation to protein synthesis, cell proliferation and cancer. Magnesium, 8, 31—44. 51. Tucker.R.W., Meade-Cobun,K. and Ferris.D. (1990) Cell shape and increased free cytosolic calcium [Ca+2]i induced by growth factors. Cell Calcium, 11, 201-209. 52. Braughler,J.M. (1987) Calcium and lipid peroxidation. Upjohn Symposium/Oxygen Radicals. The Upjohn Co. Kalamazoo MI, pp. 99—104. 53. Thompson,L.U. (1989) Nutritional and physiological effects of phytic acid. In KinsellaJ.E. and Soucie.W. (eds), Food Proteins, AOCS, Champaign.IL, pp. 410-431. 54. Thompson,H.J., Kennedy.K., Witt,M. and Jusefyk J. (1991) Effect of dietary iron deficiency or excess on the induction of mammary carcinogenesis by 1-methyl-l-nitrosourea. Carcinogenesis, 12, 111-114. 55. Southon.S., Livesey.G., GeeJ. and Johnson.I.T. (1985) Intestine cellular proliferation and protein synthesis in Zn-deficient rats. Br. J. Nutr., 53, 595-602. 56. Thompson,L.U. (1988) Antinutrients and blood glucose. Food Techno!., 42, 123-132. 57. Thompson,L.U., Button.C.L. and Jenkins,D.J.A. (1987) Phytic acid and calcium affects starch digestibility and blood glucose response in humans. Am. J. Clin. Nutr., 46, 467-473. 58. SandbergAS. and Andersson.H. (1988) Effect of dietary phytase on the digestion of phytate in the stomach and small intestine of humans. J. Nutr., 118, 469-473. 59. Anonymous (1989) Phytase and phytate degradation in humans. Nutr. Rev., 47, 155-157. 60. Spivey-Fox,M.R. and Tao.S.H. (1989) Antinutritive effects of phytate and other phosphorylated derivatives. In HancockJ. (ed.), Nutritional Toxicology. Academic Press, New York, Vol. 3, pp. 59-96. Received on March 4, 1991; revised on August 16, 1991; accepted on August 21, 1991
2045
Downloaded from http://carcin.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 22, 2015
intestinal cancer in F-344 rats by inositol hexaphosphate. Carcinogenesis, 9, 577-580. 10. Shamsuddin.A.M. and Ullah.A. (1989) Inositol hexaphosphate inhibits large intestinal cancer in F-344 rats 5 months following induction by azoxymethane. Carcinogenesis, 10, 625—626. 11. Shamsuddin.A.M., Ullah.A. and Chakvarthy.A. (1989) Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation CD-I mice. Carcinogenesis, 10, 1461-1463. 12. Baten.A., Ullah.A., Tomazic.V.J. and Shamsuddin.A.M. (1989) Inositol phosphate induced enhancement of natural killer cell activity correlates with tumor suppressions. Carcinogenesis, 10, 1595-1598. 13. Ullah,A. and Shamsuddin.A.M. (1990) Dose-dependent inhibition of large intestinal cancer by inositol hexaphosphate in F344 rats. Carcinogenesis, 11, 2219-2222. 14. Jariwalla.R.J., Sabin.R., Lawson.S., Bloch,D.A., Prender,M., Andrews.V. and Herman,Z.S. (1988) Effect of dietary phytic acid on the incidence and growth rate of tumors promoted in Fisher rats by Mg supplement. Nutr. Res., 8, 813-827. 15. American Institute of Nutrition (1977) Report of the Ad Hoc Committee on standards of nutritional studies. J. Nutr., 107, 1340—1348. 16. Bindra.G., Gibson,R. and Thompson,L.U. (1986) [Phytate][Ca/Zn] molar ratios in Asian immigrant lacto-ovo vegetarian diets and their relationship to Zn nutnture. Nutr. Res., 6, 475-483. 17. Bird,R.P. and Stamp.D. (1986) Effect of a high fat diet on the proliferative indices of murine colonic epithelium. Cancer Lett., 31, 61—67. 18. Siegers.C.P., Buman,D., Baretton.G. and Younes.M. (1988) Dietary iron enhances tumor rate in dimethylhydrazine-induced colon carcinogenesis in mice. Cancer Lett., 41, 251-256. 19. Nelson,R.L., Yoo.S.J., TanureJ.C, Andrianopoulos,G. and Misumi,A. (1989) The effect iron on experimental colorectal carcinogenesis. AnriCancer Res., 9, 1477-1482 20 McSherry.C.S., Cohen.B.I., Bokkenheuser.Y.D., Mosbach,E.H., WinterJ., Matoba,N. and ScholesJ. (1989) Effect of calcium, and bile acid feeding on colon tumors in the rat. Cancer Res., 49, 5039-6043 21. Kaup.S.M., Behling.A.R., Choquette,L.L. and Greger.J.L. (1989) Colon tumor development in DMH-initiated rats fed varying levels of calcium and butterfat. FASEB J., 3, A469. 22. Behling,A.R., Kaup,S., Choquette.L.L. and GregorJ.L. (1990) Lipid absorption and intestinal tumor incidence in rats fed varying levels of calcium and butterfat. Br. J. Nutr., 64, 505-513. 23. Zhang,L., Bird.R.P. and Bruce,W.R. (1987) Proliferative activity of munne mammary epithelium as affected by dietary fat and calcium. Cancer Res., 47, 4905-4908. 24. Sharkey.S.M. and Bruce.W.R. (1986) Quantitation of nuclear aberration as a screen for agents damaging to mammary epithelium. Carcinogenesis, 7, 1991-1995. 25. Russo,I.H. and Russo,J (1978) Developmental stage of rat mammary gland as determinant of its susceptibility to 7,12 dimethylbenzanthracene. J. Nail. Cancer Inst., 61, 1439-1449. 26. Russo.I H. and RussoJ. (1987) Biology of disease: biological and molecular basis of mammary carcinogenesis. Lab. Invest., 57, 112-137. 27. RussoJ.H., Gusterson,A., Rogers,A.E, Russo.I.H., Wellings,S.R. and van Switen.M.J. (1990) Biology of disease: comparative study of human and rat mammary tumorigenesis. Lab. Invest., 62, 244—278. 28. Lipkin.M., Vehara,K., Winawar,S., Sanchez,A., Bauer.C, Lynch,H., Blattner,W. and Faumeni J. (1985) Seventh Day Adventist vegetarians have quiescent proliferative activity in colonic mucosa. Cancer Lett., 26, 139-144. 29. Lipkin.M (1987) Biomarkers of increased susceptibility to gastrointestinal cancer. The development and application to studies of cancer prevention. Gastroenterology, 92, 1083-1086. 30. Romagnoli.P., Filippino,F., Bandettini,L. and Brugnola,D. (1984) Increase of mitotic activity in the colonic mucosa of patients with colorectal cancer. Dis. Colon Rectum, 27, 305-308. 31. Deschner.E.E. and Maskens.A.P. (1982) Significance of the labelling index and labelling index distribution as kinetic parameters in colorectal mucosa of cancer patients and DMH treated animals. Cancer, 50, 1136- 1141. 32.Sugarbaker,P.H., MacdonaldJ.S. and Gunderson.L.L. (1982) Colorectal cancer. In DeVita.V.T., Jr, Hellman.S. and Rosenberg,S.A. (eds), Cancer: Principles and Practices of Oncology. Lippincott, Toronto, pp. 643—676. 33. Stevens.R.G., Jones,D.Y., Mocozzi.M.S. andTaylor.P.R. (1988) Body iron stores and the risk of cancer. New Engl. J. Med., 319, 1047-1052. 34. Graf.E., MahoneyJ.R., Bryant.R.G. and Eaton J.W. (1984) Iron catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site. J. Biol Chem., 259, 3620-3624. 35. Bannister J.V., Barmister.W.H. and Thomally.PJ. (1984) The effect of ferritin iron loading on hydroxyl radical production. Life Chem Rep., 2, 64-72. 36. Carlin,G. and Djursater,R. (1984) Xanthine oxidase induced depolymerisa-
