Mark Messina, *Stephen Barnes

Since the initial recognition that diet plays a role in the etiology of certain cancers, particularly cancers of the breast and colon, considerable progress has been made in identifying dietary patterns associated with cancer risk. There is general agreement that high-fat, low-fiber diet, like that consumed by much of the industrialized world, increases cancer risk and that plant-based diets, rich in whole grains, legumes, and fruits and vegetables, are protective. It has been, however, considerably more difficult to identify specific foods, types of food, or components of foods that influence cancer risk.

The recent workshop on The Role of Soy Products in Cancer Prevention, sponsored by the National Cancer Institute, had two objectives: 1) to evaluate the role of soybeans, food products derived from soybeans, and specific components of soybeans in the dietary prevention of cancer and 2) to recommend research initiatives and approaches for further studies of the effect of soy intake on human cancer risk. The meeting was chaired by Stephen Barnes and organized by Mark Messina.

Isoflavones in Cancer Prevention

Kenneth Setchell, Donna Baird, and Barnes discussed the potential role of isoflavones in the prevention of cancer, Setchell reviewed the history of phytoestrogens (1), noting that plants were first observed to induce estrus in animals in 1926. Over 300 plants are now known to possess estrogenic activity (2,3). In 1946, the infertility observed in Australian sheep that grazed on a certain type of suberranean clover was attributed to the high isoflavone content of this plant (4). Ruminal bacteria in these animals convert plant isoflavones into the mammalian isoflavone equol, which, following absorption, may suppress the pituitary gonadotropic axis. Equol, a weak estrogen possessing about 0.2% of the biological activity of estrogenic effects of soybean isoflavones was stimulated coincidentally. He discovered that the soy component of diets fed to captive cheetahs, which was added for economic reasons, was responsible for the severe breeding problems in these animals (6,7).

Setchell noted that isoflavone metabolism has been studied in humans, although only superficially. In one study, sugjects fed 40g of soy daily were found to have urinary levels of equol as much as 1000-fold higher than baseline values (8,9). The low levels of urinary equol in two of the six subjects in this study indicate that the intestinal microflora (10) participate in isoflavone metabolism and that isoflavone undergo enterohepatic circulation (10). Improved analytical methods (11,12) have led to the realization that equol represents only a small fraction of the total amount of isoflavone in urine and that conjugates of the soybean isoflavones daidzein and genistein are the major forms present. The high levels of isoflavone in urine in subjects fed soy suggest that these compounds are likely to elicit a biological response (13).

Setchell conclued his presentation with a reminder (a) that all weak estrogens can also have antiestrogenic activity; (b) that tamoxifen, which has been used therapeutically for beast cancer, is structurally related to some of the phytoestrogens; and (c) that vegetarians, who may have a lower risk of certain cancers, excrete higher levels of phytoestrogens. These findings have led to collaborative studies by Barnes, Setchell, and associates (14), who used an animal model designed to test the hypothesis that phytoestrogens have a role in reduction of breast cancer risk.

Barnes began by observing that Oriental women, who have low incidence rates of breast cancer (15), consume larger amounts of soy products than do most American women. However, although fertility and reproduction in animals are adversely affected by ingestion of plant isoflavones, the amount of isoflavones in soy products consumed by Oriental women does not appear to affect their reproductive capacity.

Barnes discussed the recent animal study conducted in collaboration with Setchell and other investigators (14). In the study, consumption of soybeans significantly decreased chemically induced rodent mammary cancer. Rats were fed one of four soy products: powdered soybean chips consisting of unpurified soybeans, both raw and autoclaved; soy protein isolate composed of 91% protein; soy molasses, a concentrate of the aqueous alcohol extract of soy flour; and aqueous alcohol-extracted soy protein concentrate. All diets were isocaloric and isonitrogenous and produced similar weight gain among the animal groups throughout the study.

The first three products, all of which are rich in isoflavones, inhibited mammary tumorigenesis induced by 7,12-dimethylbenz[a]anthracene or methylnitrosourea, while the aqueous alcohol-extracted soy protein concentrate, which had a low content of isoflavones, did not. Whether the soybeans were raw or cooked made no difference in the degree of inhibition of mammary cancer; cooked soybeans were shown to be devoid of protease inhibitor activity.

Barnes said the reduction in levels of mammary tumor estrogen receptors induced by the powdered soybean chips paralleled the inhibition of tumorigenesis and supported the hypothesis that the isoflavones exerted an antiestrogenic effect. Interestingly, however, this was not the case for the soy protein isolate. The decrease in levels of mammary tumor estrogen receptors was smaller than predicted from the degree of tumor inhibition, he said, suggesting that the antiestrogenic effect of isoflavones may not be the primary mechanism responsible for inhibition of tumorigenesis. Therefore, Barnes concluded, the anticarcinogenic activity of isoflavones may not be limited to tumors containing a functional steroid receptor system. Alternative mechanisms of action may include inhibition of the activity of tyrosine protein kinases (eg, epidermal growth factor receptor tyrosine kinase) (16), DNA topoisomerase II (17), and ribosomal S6 kinase (18), as well as induction of specific cytochrome P450s (19).

Baird, before describing her recent study of the effects of feeding soy to postmenopausal women (manuscript in preparation), cited the concern of the National Institute of Environmental Health Sciences about the possible effects of low-level environmental estrogens on health. In her study, changes in estrogenic activity in postmenopausal women consuming soy over a 4-week period were examined. Soy was chosen for this study because of its high estrogenic activity (20,21), its increasing use in the United States, and the variety of products derived from soy and because soy consumption would not adversely affect nutritional status (22). Subjects consumed daily one main soy dish (1/2 cup of soybeans or 38g of texturized vegetable protein) and two soy snacks - either soy chips (a roasted soybean product) or a spread for crackers made from the whole soybean. The estimated isoflavones content was about 200 mg/day, the equivalent of about 0.3 mg/day of conjugated steroidal estrogen, assuming that the estrogenic activity of phytoestrogens is about 0.1% that of conjugated estorgen.

Baird said preliminary findings indicate that, compared with control subjects, significantly more women fed soy exhibited an estrogenic response, as demostrated by an increase in the number of superficial cells of the vaginal epithelium. She remarked that postmenopausal women were chosen for this study because of the decision to examin the estrogenic rather than the antiestrogenic effects of plant phytoestrogens. In premenopausal women with relatively high estrogen levels, the antiestrogenic effects of soybeans may have been observed.

Protease Inhibitors

Ann Kennedy, David Brandon, and Irvin Liener focused their attention on the soybean protease inhibitors. Kennedy reviewed her work, as well as that of others, in the field of protease inhibitors and cancer prevention. She noted that the soybean-derived Bowman-Birk protease inhibitor (BBI) either inhibits or prevents development of experimentally induced colon (23), oral (24), lung (25), liver (23), and esophageal cancers (von Hofe E, Newberne P, Kenned A: unpublished observations). Protease inhibitors, at the levels used in these studies, do not adversely affect animal growth. Kennedy noted that the anticarcinogenic effect of the BBI is thought to stem from its ability to inhibit chymotrypsin activity (27). She said in vitro work indicates that protease inhibitors prevent conversion of normal cells to the malignant state even at very late stages in carcinogenesis but that they have no effect on cancerous cells (28). Protease inhibitors are unique in that they cause an irreversible suppressive effect on the carcinogenic process. They have also been shown to suppress oncogene expression and to inhibit carcinogen-induced protease activity (29).

Kennedy said recent data suggest that the antigrowth effects of raw soybeans commonly attributed to protease inhibitors may actually be due to an unidentified factor(s) (30). Furthermore, in human populations consuming soybeans, the connection between pancreatic enlargement and protease inhibitors observed in animals has not been seen. In fact, incidence of pancreatic cancer is decreased in these groups (31). Kennedy noted that in vitro comparisons of the pure BBI with an extract of soybeans containing BBI indicate that the activity of the soybean extract could be directly attributable to BBI (26). However, she said an in vivo study suggests that the extract may contain an additional anticarcinogenic agent working in conjunction with the BBI (26). The extract contains approximately 50% protease inhibitor; the remaining content is unknown, but it may include isoflavones as well as other potential anticarcinogens. Kennedy commented that the lowest effective dietary levels of protease inhibitors used in these animal studies (0.1%) could be achieved by humans by modifying the diet to include soy products.

Brandon discussed the measurement of protease inhibitors in soybeans and soy products, noting the concern of the Agricultural Research Service of the United States Department of Agriculture (USDA) over the possible adverse effects of protease inhibitor intake in humans, particularly in infants (32). Enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies, have been developed for the measurement of two different protease inihibitors found in soybeans - BBI and Kunitz trypsin inhibitor (KTI) (33, 34). These procedures are suitable for quantifying residual protease inhibitor levels in foods. A variety of processed soy products, a series of soybean flours derived from seeds in the USDA Soybean Germplasm Collection, and the soybean isoline L81-4590 (lacking KTI) (35) have been analyzed. Brandon noted that an important observation from the ELISA analysis of heat-treated soy flours derived from the isoline was that KTI, not BBI, is responsible for the heat-stable activity of commercial soy flour that inhibits trypsin activity (36, 37). The microenvironment of the soy flour appears to promote heat inactivation of BBI to a greater extent than it affects KTI. This finding contrasts with the results of work showing that BBI is relatively heat stable in the pure form (38). Moisture, fat content, the presence of agents that influence changes in disulfide bonds, and itneractions with other constituents, such as carbohydrates, appear to influence the denaturation of inhibitors (39).

Brandon said analysis of infant formula has revealed that active KTI and BBI, when measured on the basis of weight per gram of protein, are reduced to about 0.1% of their levels in raw soy (40). An infant on a diet consisting exclusively of soy formula would consume about 10 mg of active KTI plus BBI per day. In toasted (autoclaved) soy flour, 20-30% of the KTI activity remains, while all of the BBI is inactivated. Analysis of tofu (soybean curd) has revealed that the protease inhibitor content varied significantly among the samples, from 4 to 30 ug of BBI and from 5 to 16 ug of KTI per gram of product. The protease inhibitor content of several soy protein isolates also varied, as much as 20-fold. Not unexpectedly, there was also a wide variation in the protease inhibitor content among varieties of soybeans. Brandon suggested that food-processing strategies could be combined with genetic approaches to optimize the protease inhibitor content of soy products.

Liener reviewed research on the potential adverse effects of consuming protease inhibitors, first noting that most work has been done with small experimental animals (41). Consumption of raw soybeans has two major effects: growth inhibition and pancreatic enlargement. Rats consuming raw soy flour for extended periods develop adenomatous nodules involving acinar cells of the pancreas (42). Additionally, raw soy flour consumption potentiates the effect of pancreatic carcinogens (43). In a study by Liener et al (44), heat treatment of raw soybeans almost completely eliminated this potentiation, while the addition of protease inhibitors to the heated product restored most of the pancreatic enlargement observed with raw soy, suggesting that protease inhibitors are at least partly responsible for pancreatic enlargement.

Liener noted that the varied response to raw soy flour among species is particularly important. Rats, mice, chickens, hamsters, and young, growing guinea pigs all exhibit pancreatic enlargement in response to protease inhibitors, while dogs, pigs, calves, and monkeys do not (45). Growth inhibition induced by soybean products is thought to result from a deficiency of the sulfur-containing amino acids caused by the dramatic increases in fecal levels of endogenous protease enzymes, particularly trypsin and chymotrypsin, two enzymes that are rich in these amino acids (46).

Commenting that pancreatic enlargement apparently stems from elevated serum levels of the hormone cholecystokinin. Liener commented that pancreatic enzyme secretion is inversely related to the level of trypsin in the intestine, a process regulated by cholecystokinin. This hormone stimulates the pancreas to produce trypsinogen, but because the protease inhibitors combine with trypsin, the suppressive effect of trypsin on intestinal release of cholecystokinin is eliminated (47).

Liener raised the question: Can the effects of protease inhibitors in small animals be extrapolated to humans? A negative feedback system in humans has been observed (48). Directly supplying BBI or raw soy flour to the duodenum causes an increase in secretion of pancreatic enzymes (48) and in blood levels of cholecystokinin (49). (BBI, in contrast to KTI, survives in gastric juice.) Despite these observations, he said, it is not possible at this time to accurately assess the health consequences of consuming processed soy products.

Phytosterols and Saponins

A. Venket Rao presented evidence for reduction of colon cancer risk by phytosterols and saponins. Both substances are common constituents of plants, but the concentration in soybeans is particularly high. Phytosterols are structurally similar to the animal sterol cholestoral. They inhibit cholesterol absorption and are almost quantitatively recoverable in fecal material, indicating that very little intestinal absorption occurs (50). Soybeans are a major contributor of phytosterols to the diet, particularly B-sitosterol (90 mg/100 g edible portion of the soybean) (51). Soybean oil is potentially an important source of phytosterols, but upon refinement and hydrogenation, phytosterol levels are reduced from 315 mg to 217 mg and 132 mg, respectively, per 100 g of oil (51). Dietary phytosterol intake among populations differs dramatically; the typical western diet contains about 80 mg/day, while Japanese and vegetarian diets provide about 400 and 345 mg/day, respectively (52, 53).

In addition to the phytosterols, whole soybeans contain significant amounts of saponins, about 5% of dry weight (54), while tofu contains approximately half that much. Saponins are amphophilic compounds having surfactant properties and, like phytosterols, bind to cholesterol and bile acids.

Rao said that while nutritional interest in both phytosterols and saponins has focused on their cholesterol-lowering properties, some data suggest that these compounds may be anticarcinogens. In rats, B-sitosterol-supplemented diets (0.2% by weight) inhibit chemically induced colon cancer (55), and phytosterols reduce, in a dose-dependent fashion, cholic acid-induced colon cell proliferation and mitotic activity (56). Diets containing phytosterols at 1% by weight are well tolerated by experimental animals (57). Dietary saponins from soybeans and other sources have been shown to enhance immunity (58, 59), are cytotoxic to Sarcoma 37 cells (60), inhibit DNA synthesis in tumor cells (61), decrease the growth of human epidermoid carcinoma cells (62) and human cervical carcinoma cells (63), and inhibit Epstein-Barr virus genome expression (64). Saponin-supplemented diets (1% by weight) as is the case for the phytosterols, normalize abnormal colonic cell proliferative activity induced by carcinogens (Rao AV: unpublished observations).

Inositol Hexaphosphate

Ernst Graf discussed the rationale for the hypothesis in which inositol-I.2,3.4,5,6-hexaphosphate (IP), not fiber, is postulated to be responsible for the inverse correlation between the incidence of colon cancer and the consumption of fiber-rich foods (65). When the [content of cereals, fruits, and vegetables is considered, the international data suggest that there is a greater negative correlation between IP and colon cancer incidence than between fiber and colon cancer incidence. lP is found in a variety of plant foods, particularly cereals, but soybeans are an especially rich source, containing about .4% on a dry-weight basis (66).

Graf noted that most nutritional interest thus far has focused on the inhibitory effect of IP on mineral absorption. IP forms tight chelates with a variety of polyvalent metals such as calcium, zinc, and iron (66). However, he said, the ability to bind metal ions, particularly iron, may provide the basis for the anticarcinogenic effects of this compound. Graf commented that iron may be a key factor, via the Haber-Weiss reaction, in the production of hydroxyl radicals, which are postulated to play a role in the etiology of some cancers (67). 1P has been shown to limit the oxidant reactivity of transition metals (66), to inhibit lipid peroxidation (67), and to inhibit experimentally induced colon cancer (68-73). It has also been suggested that 1P through absorption following dephosphorylation to 1P could be an important second messenger involved in the regulation of cell differentiation (73).

Phytochemical Variation

James Duke discussed phytochemical variation in soybeans. Duke started by noting that there alt over l0 named or numbered varieties of the common soybean Glycine max L. In these varieties, as one might expect, lies tremendous chemical variation. The genus Glvcine was originally applied to a distant relative, now known as Apios americana, which is an edible root with more protein than is found in potato (74).

The isoflavone content of soybeans varies tremendously ac cording to the plant part, variety. year harvested, and geographic location (75). Soybean hulls contain only relatively minor amounts of isoflavones, the majority of which occur in the hypocotyl, although one common isoflavone, genistein, is found primarily in the cotyledon (75). Equally significant are the reported differences in isoflavone content according to the varieties of soybeans and the year harvested. One study (75) reported a threefold variation in total isotlavone content among four varieties of soybeans, while a 30% variation was noted in a single variety of soybeans over a 4-year period. The content of individual isoflavones varied as much as 50%. Not surprisingly, location influences isoflavone content, even within fairly close geographical areas.

Duke noted that chemical variation is not limited to the isoflavones. In some instances as much as a fivetold variation was found among different phenolic acids in soybeans, many of which have also been investigated as potential anticarcinogens.

Isoflavones in Plant Physiology

Renee Kosslak described the role of isoflavones in defense strategies utilized by plants. Plants produce a wide range of products or secondary metabolites thought to enhance their survival (76) The isoflavones daidzein and genistein are the major inducers of the nodulation genes in Bradvrhizobium bacteria, which form nodules on soybeans (77).

The genetic regulation of isoflavone synthesis in plants is not well understood, in part because of the limited number of appropriate mutants affecting this pathway (78.79). In soybeans, near-isogenic lines that differ in their root fluorescence are being examined to determine whether they are active in genetic regulation of isoflavone synthesis (80). These differences in root fluorescence In soybeans were first observed in 1934.) There are five loci that affect root fluorescence (80), and al though specific substances responsible for this property have not been identified, isoflavones are thought to be involved. Preliminary data indicate that the levels of daidzein, genistein, and coumestrol, which is also a phytoestrogen, were either reduced or absent in root extracts from three of the nonfluorescent isolines tested (Kosslak R: unpublished observations).

Kosslak suggested that if future research implicates ‘so flavones and/or phytoestrogens as important dietary factors in cancer prevention and if the demand for soybean specialty products materializes, it may be possible to manipulate levels of these compounds in soybeans, using root fluorescence as a marker.

Soybean Processing

Bernie Szuhaj briefly discussed soybean processing procedures (81-83). Solvent extraction is the primary method of producing soybean products today, Soybeans entering the plant are first cleaned, cracked, and dehulled. Then moisture is added so they can be “flaked,” leaving a product that is 3% hypocotyl, 89% cotyledon, and 8% hulls. The oil is removed from the flakes by hexane, producing defatted flakes and soybean oil. From the defatted flakes come a variety of products with a protein content, on a dry-weight basis, that ranges from about 50% for soy flour and grits to about 60% for protein concentrates and about 90% for protein isolates. The primary difference between soy protein concentrates and isolates is the larger percentage of carbohydrate in the soy protein concentrates Many commercial doughnuts contain soy flour, and, in Europe and Asia, there is particular interest in the use of full- fat soy flours for baking.

Szuhaj noted that most soybean production today goes into animal feed, while the soy protein concentrates and isolates are marketed primarily for their multifunctional properties, such as emulsifying gelling, fat-binding, texturizing, and dough forming. Soy products play a major role in the food chain. They are added to a wide variety of foods, from cereals to chili. Some meat products, such as ground beef, contain up to 25% soy. These products have been used in the Aimed Forces’ canteens since (983 and in the federal school lunch program.

Discussion

This workshop had two objectives: I to evaluate the relation ship between the risk of certain cancers and consumption of soybeans, products derived from soybeans, and/or specific components of soybeans and 2) to recommend research initiatives aimed at clarifying this relationship. The consensus of the meeting was that there are sufficient data to justify studying the impact of soybean intake on cancer risk in humans.

There were three workshop recommendations. First, future dietary studies involving soybeans should be carried out using soy products rather than isolated compounds, since soybeans appear to contain several potential anticarcinogens. Additionally, because components of food interact, both negatively and positively, with each other, the potential benefit of soy products can not be accurately predicted solely on the basis of the effects of individual soybean components. This does not, however, prohibit future use of isolated soybean components as chemo-preventive agents in clinical trials. Second, standardized and improved analytical methods are needed so that the contents of all soy-based materials employed in soybean research, whether soybean fractions or soy products, can be accurately described. This methodology will allow for valid comparisons among studies. Third, basic research on the absorption, metabolism, and physiology of potential anticarcinogens in humans should be conducted. This research will likely help to determine the clinical relevancy of these compounds and to provide a basis for selecting specific soy products for use in future dietary studies.

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