American Health Foundation, 1 Dana Road, Valhalla, NY 20595 and
1 Kraft Foods Inc., Glenview, IL 60025, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: DMBA, dimethylbenz[a]anthracene; IDSP, isoflavone-depleted soy protein; NMU, N-nitroso-N-methylurea; SP, intact soy protein.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies in human populations relating soy consumption to reduced breast cancer risk have produced conflicting results (1,19). A recent paper by Petrakis et al. (20) reported that prolonged consumption of soy protein isolate by a group of healthy US women exerted a stimulatory effect on the pre-menopausal female breast (i.e. increased hyperplastic epithelial cells in duct fluid aspirates), which the authors attributed to the estrogenic effects of isoflavones. Published studies on the preventive effect of soy or soy isoflavones in mammary tumor models have also proven inconsistent (1,5,6,21,38). The precise reasons for this are unclear, but they include the use of varying types of soy protein isolates, purified genistein, different tumor models and a variety of basal diets and experimental protocols.
The purpose of the present study was to determine, in a well-established mammary tumor model, whether the putative chemopreventive effect of soy protein was attributable to the soy protein per se or to the isoflavone fraction of soy protein. To this end, N-nitroso-N-methylurea (NMU)-treated animals were fed two levels of intact soy protein (SP) or two levels of alcohol-washed isoflavone-depleted soy protein (IDSP) and mammary tumor yields were compared with controls fed the casein-based AIN-93G diet.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protocol for experimental mammary tumor induction
One hundred and fifty virgin female F-344 rats (starting age 28 days old; Charles River, Raleigh, NC) were maintained on the standard Open Formula Rat and Mouse Ration (NIH-07) diet (4.5% fat, 23.5% protein, 50% carbohydrate and 4.5% fiber) (Zeigler Bros., Gardners, PA) until 43 days of age, when they were placed on the experimental diets. All rats were then assigned to one of five experimental groups of 30 animals each by recognized randomization procedures (25) to equalize initial weight. The five groups were as follows: group 1, 20% SP; group 2, 10% SP; group 3, 20% IDSP; group 4, 10% IDSP; group 5, control. At 50 days of age, all rats received a single dose (40 mg/kg body wt) of NMU (Ash Stevens Inc., Detroit, MI) by tail vein injection. NMU was dissolved in a few drops of 3% acetic acid and diluted with distilled water to give a stock solution of 10 mg NMU/ml, which was administered within 2 h of preparation (22).
Soy protein
Soy protein isolate (Supro 670HG lot no. C6L-XRM-004) containing intact and alcohol-washed soy protein with trace isoflavones (Supro 670 IF lot no. P6J-XRP-9001) were supplied by Protein Technologies Inc. (St Louis, MO). SP was composed of 87% protein, 4% water and 4.8% fat; IDSP, 93% protein, 5% moisture and 1.2% fat. Phytate levels of 11.5% were present in both SP and IDSP. Endogenous protease inhibitors present in soy were heat-inactivated during processing of the protein isolate. Saponins, which are present at low levels in soy protein isolates, were reported to be reduced by half during the alcohol washing procedure (personal communication from Protein Technologies Inc.).
According to data provided by Protein Technologies, isoflavonoids were present in both conjugated and unconjugated (aglycone) form, ~50% aglycone and 50% conjugated. The total concentration (conjugated and unconjugated) (w/w) of isoflavonoids in the SP isolate was 2.89 mg/g isolate; the concentration of total isoflavonoids minus glucuronide and sulfate groups was 1.67 mg/g isolate. The total concentration of genistein (free and conjugated) was 1.07 mg/g; that of daidzein was 0.52 mg/g. The IDSP isolate contained only trace amounts of isoflavones: total (conjugated and free) isoflavones was 0.20 mg/g isolate. The weight of isoflavones minus glucuronide and sulfate groups was 0.12 mg/g and total genistein and daidzein concentrations were 0.07 and 0.04 mg/g, respectively. Glycetein was present at low levels in both soy isolates: 0.15 (SP) and 0.01 mg/g (IDSP).
Total (aglycone) isoflavone levels in soy protein isolates were measured in our laboratory after acid hydrolysis. Genistein levels were 1.06 mg/g isolate in the SP sample and 0.07 mg/g in the IDSP sample; daidzein levels were 0.54 and 0.05 mg/g isolate in the SP and IDSP samples, respectively. These values were in close concordance with those provided by Protein Technologies Inc. using a different HPLC method which measured both glycones and aglycones without prior acid hydrolysis.
Diets
The diet used was a modified AIN-93G purified rodent diet (23), with the addition of both L-methionine and L-cystine. Basal mixes were obtained commercially from Dyets Inc. (Bethlehem, PA). The milk protein casein in the AIN-93G diet (20% w/w) was totally or partially replaced in the diets containing SP or IDSP. Diets were prepared in our diet kitchen in 15 kg lots by mixing basal mix concentrates in a Hobart planetary mixer with specific amounts of casein, SP or IDSP to give a final protein content of 20% in all experimental diets. Soy oil was added to the IDSP diet to compensate for its lower oil content. In this manner all animals received equal amounts of soy oil. Diets containing SP and IDSP were stored at 4°C until use (Table I). Rats were fed three times per week and feeders were removed and washed after each feeding. The rats were fed the experimental diets 1 week prior to NMU administration and remained on these diets for the duration of the experiment (18 weeks).
|
Urine collection, necropsy and histopathology
Approximately 18 weeks after NMU administration, the experiment was terminated. One week prior to termination, three rats from each group were placed in metabolism cages and urine collected over a 24 h period after a 1 day acclimation period. Urine, typically 1015 ml/24 h, was collected on dry ice in 50 ml conical centrifuge tubes then thawed and centrifuged at 200 g for 20 min at 4°C. The clear supernatant was decanted and stored at 20°C. At study termination rats were killed by carbon dioxide asphyxiation and mammary tumors (classified as palpable or non-palpable but grossly visible) were excised and the two largest diameters measured with vernier calipers. The tumors were then fixed in 10% buffered formalin, embedded in paraffin blocks and stained with hematoxylin and eosin for histological examination. Histological diagnosis of mammary tumors was based on criteria outlined by Young and Hallowes (24) and Russo et al. (25).
Statistical analysis
Tumor-free survival was estimated separately for each group by the KaplanMeier product limit estimate for censored data (26). The survival distributions for the high and low dose for each treatment group were then compared with controls by the log rank test (27,28). The purpose of the analysis was to test the null hypothesis that the survival distributions of all those groups were equal. In addition to the overall test of significance, pairwise comparisons between groups were made.
Tumor incidence (expressed as the percentage of tumor-bearing animals) was compared among the groups by Fisher's exact test. Overall dose-related associations between tumor incidence and the two SP or IDSP doses were tested by use of Armitage's test for linear trends in proportions (29,30). Tumor multiplicity among the groups (categorized as 0, 12, 34 or >5 tumors/rat) was compared by use of the MantelHaenszel (31) 2 test. In addition, overall dose-related associations between tumor multiplicity in high and low dose treatment groups were tested by linear regression analysis. In addition, tumor volume and multiplicity were compared for each treatment group using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test (32).
Tumor volumes were assessed by applying the formula: vol (cm3) = D12xD2, with D1 representing the largest tumor diameter and D2 the next largest tumor diameter in centimeters.
The overall weight gains in the animals of all groups were compared by use of single classification ANOVA with repeated measures followed by Dunnett's test (32,33). The test of interest was the interaction between weight and time to evaluate the null hypothesis of no difference in weight gain over time among the groups. Pairwise comparisons among the groups were also conducted.
All statistical tests were two-tailed and were considered statistically significant at P < 0.05. Significance tests for all pairwise comparisons were adjusted for multiple comparisons by multiplying the actual P value by the number of comparisons made (in this case two) for the evaluation of statistical significance.
Isoflavone extraction from soy protein isolate and urine
For soy protein total isoflavone determinations, a 0.51.0 g sample was taken and a mixture of 10 ml 10 M HCl, 40 ml 96% ethanol (containing 0.05% butylated hydroxytoluene) and 20 p.p.m. flavone, as internal standard, was added (34). The suspension was dispersed by sonication for 10 min and then refluxed for 3 h. After cooling to room temperature, ethanol was added to replace that lost during refluxing. A 1.2 ml aliquot of the reaction mixture was centrifuged at 850 g for 15 min and 20 µl of the clear supernatant was injected directly into the HPLC system.
For urinary isoflavone extraction, a two-stage enzymatic procedure was used (35). To 1 ml of a urine sample, 1 ml of 0.2 M acetate buffer (pH 4) containing 100 µl flavone (400 µg/ml in 96% ethanol) was added as internal standard. (For animals fed IDSP, 2 ml of urine was used.) The resulting mixture was then filtered through a solid phase C18 reversed phase extraction column (PGC Scientific, Gaithersburg, MD) preconditioned with 5 ml methanol and 5 ml acetate buffer (pH 4). The isoflavone fraction was then eluted with 6 ml 100% methanol and the eluant dried under N2 gas. The dried residue was dissolved in 1 ml of 0.2 M phosphate buffer (pH 7.0) to which was added 50 µl ß-glucuronidase (isolated from Escherichia coli 200 kU/l) and 50 µl of arylsulfatase (isolated from Helix pomatia, 15 kU/l; Boerhinger-Mannheim, Indianapolis, IN) and incubated for 1 h at 37°C. To terminate the reaction, 0.9 ml of 100% methanol was added to the reaction mixture. The mixture was then filtered through a syringe filter (Whatman, Clifton, NJ) and the sample, containing deconjugated isoflavones, was stored at 4°C. For analysis 20 µl of sample was injected into the HPLC system. For animals fed IDSP, further concentration was required. This was done by partitioning isoflavones into ethyl acetate (3x2 ml), drying under N2 gas and redissolving in 300 µl of mobile phase (see below), to which 100 µl of 0.2 M acetate buffer, pH 4, was added.
HPLC analyses (34,35)
Reversed phase HPLC analysis combined with UV detection was used to measure isoflavones. All HPLC analyses were carried out on a Nova Pak C18 (150x3.9 mm i.d., 4 µm particle size) reversed phase column (Waters, Milford, MA) coupled to an Absorbosphere C18 (10x4.6 mm, i.d., 5 µm particle size) guard column (Alltech, Deerfield, IL). Elution was at a flow rate of 0.8 ml/min with the following mobile phase: A, acetic acid/water (10:90 v/v); B, methanol/acetonitrile/dichloromethane (10:5:1 v/v/v). The linear elution gradient used was as described by Franke et al. (35). Analytes were detected by their absorbance at 260 nm. Analyses were conducted using a Waters model 510 HPLC pump (Milford, MA) equipped with a Waters 745 Integrator and a Shimadzu SPD-10A UV-visible detector (Kyoto, Japan).
Standard stock solutions were prepared by dissolving 2 mg crystalline genistein (5,7,4'-trihydroxy isoflavone), daidzein (7,4'-dihydroxy isoflavone) or flavone (2-phenyl-4H,1-benzpyran-4-one) (Sigma Chemical Co., St Louis, MO) in 20 µl dimethylsulfoxide followed by addition of methanol to yield a 25 M stock solution. The concentrations of the stock solutions were determined by absorbance readings using the extinction coefficients for genistein (263 nm, E37154) and daidzein (250 nm, E27542).
Standard curves for genistein and daidzein were generated from authentic standards and their concentrations in soy protein and urine samples were calculated from peak areas obtained from HPLC analyses using the slopes of the standard curves for quantitation. The retention time for genistein was 20 min, for daidzein 17 min and for flavone 33 min. Data were presented as mg genistein (daidzein)/g protein isolate and µg genistein (daidzein)/24h urine or µg/ml urine.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, in vivo studies on the chemopreventive role of isoflavones in laboratory animal models of breast cancer have yielded conflicting results. Most of these have focused on foods rich in isoflavones rather than purified isoflavones. Ten published reports have focused on soy or soy protein isolates because of the exceptionally high levels of the isoflavone genistein found in soy. Of these, three found no protective effect (3638), one found an enhancement of tumorigenesis (39), one found a weak protective effect (40) and four reported protective effects (8,9,41,42). All of these studies involved radiation or chemically induced primary mammary tumors initiated in SpragueDawley rats, with the exception of that of Gridley et al. (39), which was conducted in the `spontaneous' mouse mammary tumor model. As stated earlier, the reasons for these discrepancies are unclear. They could be due to differences in the soy matrices used, i.e. raw soybeans, soy protein isolates (high isoflavone concentrations), soy protein concentrates (low isoflavone concentration), different amounts of soy (1050%), different basal diets, (casein-based, semi-purified or grain-based) and different exposure times.
Recently Connolly et al. (7) tested the inhibitory effects of toasted soy chips in the mouse xenograft model, which permits growth of human cancer cells in a rodent. Soy had no effect on the growth of MDA-MB-431 human breast cancer cells but did inhibit their ability to metastasize to the lung, implying that isoflavones may exert an effect on the metastatic stage of breast carcinogenesis. Consistent with this finding, Scholar and Toews (43) reported that genistein inhibited invasion by murine carcinoma cells in an in vitro invasion assay.
In order to more directly test the isoflavone hypothesis, several studies have treated rats with purified genistein or daidzein (44,45). Constantinou et al. (45), for example, reported non-significant reductions in NMU- and dimethylbenz[a]anthra- cene (DMBA)-induced mammary tumors with i.p. injections of 0.8 and 0.4 µg genistein and daidzein. Recent studies by La Martiniere et al. (46,47) suggest that the timing of exposure to hormonally active chemicals, such as genistein, could influence the outcome of tumor bioassays. It was reported that rats exposed neonatally to genistein were less responsive to subsequent initiation by the host-activated carcinogen DMBA. This was not due to suppression of DNA adduct formation; instead it was proposed that genistein accelerated the genetically programmed differentiation of the mammary gland, thereby conferring protection against subsequent carcinogen insult. Hence, it is conceivable that soy consumption could exert subtle effects not in the adult, but on the mammary gland during gestation, the neonatal period or puberty, which may result in protection against breast cancer in later years. In sum, the aggregate data from experimental animal bioassays, while suggestive in some cases of inhibitory effects, does not provide compelling evidence in support of soy, soy protein isolates or soy isoflavones administered in the physiological range as potent chemopreventive agents in mammary carcinogenesis.
Hawrylewicz et al. (8) have suggested that the tumor-inhibiting effects of soy may be due not to the presence of isoflavones but to the fact that soy protein has half of the essential amino acid methionine as the milk protein casein. As a means of removing dietary methionine as a potential confounding variable we chose the newly recommended AIN-93G diet for this study, rather than the commonly used AIN-76A diet (23). Because soy protein is methionine-deficient compared with casein, for the present study we used a modified AIN-93 diet which includes increased levels of both L-cystine and L-methionine. In addition, the AIN-93G diet substitutes 7% soy oil for 5% corn oil. Soy oil contains linolenic acid, an n-3 fatty acid, and has a more balanced n-6/n-3 ratio (7.5) than corn oil, which is rich in n-6 fatty acids but contains only trace amounts of n-3 fatty acids.
Isoflavones are a class of non-steroidal phytoestrogens of plant origin, present as glycoconjugates. In soy protein isolates, due to chemical processing, approximately half of the isoflavones are present as glycones and the other half as aglycones (11). When glycones are ingested they undergo hydrolysis by intestinal bacteria with the release of hormone-like aglycones. The aglycones enter the circulation and are reconjugated in the liver and undergo enterohepatic recirculation. The reconjugated isoflavones are excreted in the urine as glucuronides and sulfates. In the present study, urinary output of genistein and daidzein varied from 7 to 19% of ingested isoflavones, a result consistent with that reported by Barnes et al. (4). No genistein could be detected in the urine of controls fed the AIN-93 diet. Trace levels of daidzein were found in the urine of controls. Urinary daidzein levels in soy-fed rats were similar to genistein levels despite the fact that the concentration of genistein in the soy protein isolate was twice that of daidzein. This finding is consistent with those reported by Franke et al. (35), Petrakis et al. (20) and Lu et al. (48) in human feeding studies. The reasons for the disproportionate excretion rates of the two isoflavones are unknown. Possibly genistin (the conjugated form of genistein) may be more easily hydrolyzed to genistein or more extensively metabolized in the gut or excreted to a greater extent by the fecal route.
In groups 1 and 2, fed 20 and 10% SP, there was a positive correlation between the total dose of isoflavones ingested and that excreted in the urine when expressed in terms of µg isoflavones/ml urine but not when expressed in terms of total 24 h urinary excretion. However, in groups 3 and 4, fed 20 and 10% IDSP, there was no relationship between ingested and excreted isoflavones. These results suggest that the use of urinary isoflavone concentrations as a biomarker for dietary intake may be valid in populations consuming relatively high levels of soy products but not in those consuming marginal levels.
Based on the concentration of genistein and daidzein in the experimental diets and the amount of food consumed, daily intake of isoflavones in this study ranged from 1.5 mg/rat in the 10% SP group to 3 mg/rat in the 20% SP groups. In the 10 and 20% IDSP groups daily intakes of genistein were estimated to be 0.13 and 0.23 mg/rat, respectively. In Japan, daily intake of genistin plus genistein ranges from 7.8 to 12.7 mg/person/day (1). Extrapolating from rat to human, based on an average weight of a F-344 female rat of 200 g and of a human 70 kg, the rats receiving 10 and 20% SP consumed approximately 50 and 100 times the intake of an average Japanese. On a surface area basis, assuming the average surface area of a rat to be 324 cm2 and of a human 18 000 cm2, rats consumed approximately 8 and 17 times the genistein intake of the average Japanese. Using the same ratios, rats consuming the IDSP diets were exposed to 4.5 and 9 times more isoflavones on a body weight basis and 0.7 and 1.4 times more on a surface area basis than the average Japanese. Hence, the isoflavone concentrations used in the present study are relevant to soy consumption patterns in Asian countries.
In summary, the results of this study do not support the hypothesis that phytoestrogens present in soy act to inhibit the development of chemically induced mammary tumors.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|