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Article

Dietary Soy Protein Is Associated with Reduced Intestinal Mucosal Polyamine Concentration in Male Wistar Rats¹

Weiqun Wang ^*,2 and Carl M. Higuchi^**

^* Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011; Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI 96813; and ^** John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822

²To whom correspondence should be addressed.

	ABSTRACT

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Quantitation of polyamine levels has been correlated with biomarkers of proliferation in the colon mucosa where dysregulated epithelial hyperproliferation is associated with colorectal cancer risk. This study was performed to assess the response of polyamine measurements to dietary factors in an animal model. Male Wistar rats were fed purified diet or diets substituted by 20% lard fat, 20% beet fiber and 20% soy protein. After 2 wk, mucosal polyamines were measured along intestinal tracts by HPLC. In rats fed the control diet (n = 10), mucosal polyamines were found at high levels in the duodenum, jejunum and ileum but at low levels in the cecum, colon and rectum. Compared with rats fed the control diet, those fed the 20% lard diet showed greater polyamine levels in the large intestine (P < 0.05, n = 10), but those fed the 20% fiber diet exhibited lower polyamine levels in the small intestine (P < 0.05, n = 9). However, rats fed the 20% soy protein diet had lower polyamine levels in both small and large intestines (P < 0.05, n = 15). Significant linear correlations were observed between rectal polyamine levels and the dietary energy intakes in these four diet groups (r = 0.972–0.991, P < 0.001). Supplementation of 0.1% soy isoflavones to the basal diet or 0.3% DL-methionine to the 20% soy protein diet for 4 wk did not affect polyamine levels. The results indicate that soy protein reduced mucosal polyamine levels, at least in part, through reduction of energy intakes. Further studies are warranted to verify that polyamine levels in intestinal mucosa are useful as an intermediate endpoint of the dietary risk factors.

KEY WORDS: • polyamines • soy proteins • biomarker • colorectal cancer • rats

	INTRODUCTION

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Dysregulated epithelial proliferation within the normal colonic mucosa appears to be a hallmark of increased susceptibility to neoplasia. Measures of colonic mucosal proliferation have thus been proposed as an index of colorectal cancer risk, or as surrogate endpoint biomarkers for cancer prevention trials (Lipkin 1988 , Moore and Tsuda 1998 ). Although several methods of assessing mucosal proliferation are in investigational use at present, measurement of mucosal polyamines appears to be an alternative measure of dysregulated colorectal proliferation (Higuchi and Wang 1995 , Pegg 1988 , Wang et al. 1996 ).

Polyamines are ubiquitous short-chain aliphatic amines that play an important role in cellular proliferation and differentiation (Heby 1991 , Morgan 1998 ). Cellular levels of polyamines increase significantly when cells are stimulated to proliferate. Abnormal hyperproliferative cells such as preneoplastic tissue exhibit a very high requirement for polyamines to sustain cell growth through elevated DNA, RNA and protein synthesis (Pegg 1988 ). Elevated polyamine levels have thus been found within neoplastic tissues as well as within the flat colonic mucosa of individuals at increased risk for neoplasia (Kingsnorth et al. 1984 , Wang et al. 1996 ). Furthermore, the polyamine biosynthetic enzymes, ornithine decarboxylase (ODC)³ and S-adenosylmethionine decarboxylase (SAMDC), are highly regulated in all of the cells and respond to a wide variety of growth-promoting stimuli. A link between polyamine metabolism and colorectal cancer risk seems well established in view of the reported association between colorectal mucosal ODC activity and cancer risk (Koo et al. 1988 , Luk and Baylin 1984 , McCann et al. 1992 , McGarrity et al. 1990 , Narisawa et al. 1989 ), and because polyamine biosynthesis inhibition by ODC and/or SAMDC inhibitors is protective against colorectal carcinogenesis in animal models (Li et al. 1999 , Loser et al. 1997 , Pegg et al. 1998 , Verma 1989 ) and in on-going human clinical chemopreventive trials (Meyskens and Gerner 1999 ).

Epidemiologic, animal and clinical studies have shown that dietary factors can be implicated in the etiology of colorectal cancer (Shike 1999 ). There is substantial evidence from studies in laboratory rodents that a diet high in fiber can retard carcinogenesis, but a diet high in saturated fat can promote tumor growth. Soy proteins have also been shown to inhibit the growth of various tumors including colon carcinogenesis in animal models (Hawrylewicz et al. 1995 , Messina and Barnes 1991 , Thiagarajan et al. 1998 ). It is believed that this potential protection or promotion against colon cancer could be associated with a modified cellular proliferation (Kim et al. 1998 , Lee et al. 1993 ). Because mucosal polyamine measurements have been shown to reflect cellular proliferation, it can therefore be hypothesized that altered food contents in diets and modified mucosal proliferation will correspond to changes in mucosal polyamine measurements.

To validate mucosal polyamine analyses as a quantitative biomarker of dietary risk factors experimentally, the distribution of mucosal polyamines was determined along the intestinal tracts and the response of mucosal polyamines to certain dietary healthy or unhealthy factors was measured by using an in vivo animal model. In addition, the plausible mechanisms by which soy protein reduces mucosal polyamine levels were investigated.

	MATERIALS AND METHODS

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Experimental animals.

Male Wistar rats (n = 90), 5–6 wk of age, weighing 161–203 g (Sasco, St. Louis, MO) were housed individually in raised stainless steel cages with wire-mesh floors and fronts under controlled conditions of light (12-h light:dark cycle), humidity (50 ± 15%) and temperature (22 ± 2°C). Water and food were consumed ad libitum. Body weight and water and food consumption were monitored weekly throughout both experiments.

Experimental diets.

A purified diet according to the formula given by Meyer et al. (1982) was used as a basal diet. In the fiber and fat diets, the carbohydrate pool was replaced partially by either 20% fiber (freeze-dried table beets, Beta vulgaris L.) or 20% lard (full refined lard; U.S. Biochemical, Cleveland, OH). In the soy protein diet, the expense of casein was completely replaced by 20% soy protein isolate. Soy protein isolate was prepared from dehulled and defatted soybeans by removal of most of the nonprotein components, containing about 90% protein on a moisture-free basis (Protein Technologies International, St. Louis, MO). The contents of isoflavones in these diets were measured by using our established HPLC method (Wang et al. 1994 ). The aglycone concentrations of genistein, daidzein and glycitein in the 20% soy protein diet were 164.2, 114.6 and 20.8 µg/g, respectively. The amount of isoflavones was not detectable in the basal, fiber and lard diets. The other two of the six diets used in this study contained either 0.1% isoflavones (genistein:daidzein , 1:1, ICN, Costa Mesa, CA) in the basal diet or 0.3% DL-methionine (ICN) in the 20% soy protein diet. The composition of these six diets is summarized in Table 1 . Diets were pelleted, dried and then stored at 4°C for not >1 mo.

After 1 wk acclimation to the basal diet, rats were randomly assigned into different diet groups. Two experiments were conducted. Experiment 1 included four diet groups as follows: 1) basal diet (n = 10); 2) 20% fiber diet (n = 9); 3) 20% lard diet (n = 10); and 4) 20% soy protein diet (n = 15). Experiment 2 involved four diet groups as follows: 1) basal diet (n = 8); 2) 0.1% soy isoflavone in basal diet (n = 15); 3) 20% soy protein diet (n = 8); and 4) 0.3% DL-methionine in 20% soy protein diet (n = 15). Rats were killed while under ethyl ether anesthesia after 2 wk of consuming the experimental diets in Experiment 1 or 4 wk in Experiment 2. The entire intestine was removed for mucosal samples. The protocols were approved by the University Animal Care and Use Committee.

Intestinal mucosa sample.

The entire intestine was excised and placed immediately into ice-cold 10 mmol/L PBS, pH 7.4. The whole intestine was then divided into seven segments, representing duodenum, jejunum, ileum, cecum, proximal colon, distal colon and rectum. Each segment was slit open and washed completely with chilled PBS. Duplicate mucosal samples were gently scraped off with a microscope slide from each segment and stored at -70°C until polyamine analysis.

Polyamine assay.

Each mucosal sample was suspended in ice-cold PBS and homogenized on ice using a Polytron Homogenizer (Brinkman Instruments,, Westbury, NY). After removal of gross debris by centrifugation, protein content of the supernatant was measured by a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). Diaminododecane (Dad) was added as an internal standard, and then proteins were precipitated with a final 0.5 mol/L perchloric acid. The protein-free supernatant (500 µL) was admixed with 350 µL saturated sodium carbonate and 400 µL of 37 mmol/L dansyl chloride; then the mixtures were incubated at 60°C for 1 h. Dansylated polyamines were extracted in toluene, dried, then redissolved in 100 µL of acetonitrile and finally quantified with the use of our established HPLC method (Higuchi and Wang 1995 ). Briefly, a reverse-phase chromatography procedure using a Perkin-Elmer Peco-sphere 3 x 3 CR C₁₈, 33 x 4.6 mm i.d. cartridge column with 10 mmol/L heptanesulfonate buffer, pH 3.4, in acetonitrile gradient at a flow rate of 2.5 mL/min was applied. Dansylated polyamines were detected using fluorescence detection set for excitation at 330 nm and emission at 470 nm, and the peak areas indexed to an internal standard peak area were used to calculate the values of mucosal polyamine levels. Results of this HPLC have been found to be highly reproducible, i.e., the CV of replicate measurements within a single batched assay or assayed separately on different days was consistently <6%. With the use of this method, the limit of detection is ~0.2 nmol/mg protein for acetyl spermidine (AcSpd), cadaverine (Cad), putrescine (Put), acetyl spermine (AcSpm), spermidine (Spd) or spermine (Spm).

Statistical analysis.

The SAS statistical system 6.12 (SAS Institute, Cary, NC) was used for statistical analysis. For polyamine levels, the significance of differences between diet groups was determined by one-way ANOVA and comparisons between diet groups were analyzed by Tukey’s post-hoc test. Correlations between the rectal mucosal polyamine levels and weekly energy intakes were computed by Pearson’s correlation coefficients, using mean values of each diet group as a partial correlation between groups. Analyses of diet treatment with individual energy intake for each animal as a covariant were performed by the General Linear Models procedure of SAS. Data are presented as means ± SD. Differences were considered significant at P 0.05.

	RESULTS

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During this short experiment, body weights, food consumption and energy intakes were not significantly influenced by experimental diets (Table 2 ). Although there was no significant effect, the data for weekly energy intakes revealed consistently that fat diet > basal diet > fiber diet > soy protein diet. Diet did not affect water consumption.

View larger version (26K): [in this window] [in a new window]   Figure 1. Typical polyamine chromatograms obtained with standards (A) and rat mucosa extracts (B). Polyamine standards at 6 µmol/L for both spermidine and spermine, and 1.5 µmol/L for all the other polyamines including acetyl spermidine, cadaverine, putrescine and acetyl spermine were mixed together with 3.6 µmol/L of diaminododecane as an internal standard. Rat mucosa samples were homogenized, extracted and dansylated before HPLC analysis. A reverse-phase chromatography procedure using a Perkin-Elmer Peco-sphere 3 x 3 CR C18, 33 x 4.6 mm i.d. cartridge column with 10 mmol/L, pH 3.4 heptanesulfonate buffer in acetonitrile gradient at a flow rate of 2.5 mL/min was applied. Dansylated polyamines were detected using fluorescence detection set for excitation at 330 nm and emission at 470 nm, and the peak areas indexed to internal standard peak area were used to calculate the values of mucosal polyamine levels.

Significant linear correlations were observed between average rectal polyamine levels and weekly energy intakes in these four diet groups (r = 0.972–0.991, P < 0.01; Fig. 2 ). In addition, analyses of diet treatment with individual energy intakes for each rat as a covariant showed that these significant correlations still existed after adjustment for energy intake (P < 0.05), suggesting an association of mucosal polyamine levels with dietary variables other than energy intakes.

View larger version (25K): [in this window] [in a new window]   Figure 2. Correlations between weekly energy intakes and mean levels of rectal mucosa polyamines in rats fed purified diet or diets substituted with 20% lard fat, 20% beet fiber and 20% soy protein for 2 wk. Pearson’s correlation coefficient (r) was determined (r = 0.972–0.991, P < 0.001).

	DISCUSSION

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Mucosal polyamine levels, as a measure of dysregulated colorectal hyperproliferation, have been demonstrated in our previous studies to be significantly associated with colorectal cancer risk in a human case-control study (Wang et al. 1996 ), but enthusiasm for this excellent application is tempered by concerns related to the applicability of the inconvenient sampling in human subjects. The availability of experimental studies conducted in animal models, however, may provide a unique opportunity to apply mucosal polyamine measurements as an intermediate biomarker for colorectal cancer risk.

In this study, we first measured the mucosal polyamine distribution along intestinal tracts in rats fed a normal basal diet and found a high level of mucosal polyamines in the duodenum, jejunum and ileum and a low level in the cecum, colon and rectum. This distributive profile of intestinal polyamine levels parallels the established pattern of cellular proliferation in the normal intestine, which decreases from duodenum to colon (Dowling 1982 ). This is also consistent with a previous study by Hosomi et al. (1986) who reported a similar distribution of mucosal polyamines as measured by a semiquantitative method.

We then evaluated the response of mucosal polyamines to certain dietary risk factors including fat, fiber and soy proteins. Relative to rats fed the basal diet, we observed the following: 1) mucosal polyamine levels in the large intestine were increased significantly in response to the 20% lard fat diet; 2) mucosal polyamine levels in the small bowel were decreased significantly in response to the 20% beet fiber diet; 3) mucosal polyamine levels in both large and small intestines were decreased significantly in response to the 20% soy protein diet. These corresponding changes of mucosal polyamine levels to dietary high or low risk factors may thus support a value of mucosal polyamine analysis as an intermediate index of colorectal cancer risk in dietary intervention studies. However, additional studies that include different diets and different types of nutrients are warranted to support this conclusion.

Finally, we addressed the mechanisms of polyamine levels regulated by dietary factors, especially by a soy protein diet. The significant linear correlation observed between rectal polyamine levels and dietary energy intakes in four different diet groups indicates a contributory effect of dietary energy consumption on mucosal polyamine levels. Although the detailed mechanism is not clear, the correlation of energy intake with mucosal polyamine levels is not unexpected. An increase in energy intake in rodents has been shown consistently to be associated with elevated rates of growth and metabolism, and a decrease of energy intake (energy restriction) has been demonstrated repeatedly to extend life span and inhibit spontaneous and chemically induced neoplasia (Birt et al. 1991 , Leakey et al. 1998 ). Moreover, cancer prevention by energy restriction has been suggested to be mediated mainly through reduction of cellular proliferation (Fischer and Lutz 1998 ). Therefore, the strong correlation between rectal mucosal polyamine levels and energy intakes demonstrated in this study may indicate an energy-reduced mechanism for a high soy protein diet in cancer prevention.

In addition, subsequent analyses of diet treatment with individual energy intakes for each rat as a covariant showed that a significant correlation between diet groups and rectal mucosa polyamine levels still existed after adjustment for energy intake (P < 0.05). That may indicate an association of mucosal polyamine levels with other dietary variables such as diet composition. The inhibitory effect of soy protein on carcinogenesis in animal models has been attributed to the presence of certain anticarcinogenic phytochemicals such as phytoestrogenic isoflavones (genistein and daidzein). Genistein and daidzein have been shown to exhibit a number of chemopreventive characteristics that may be associated with cancer prevention (Adlercreutz 1995 ). The total isoflavone aglycones in the 20% soy protein diet were ~0.03%. Supplementation of soy isoflavones up to 0.1% in the basal diet, however, did not significantly affect mucosal polyamine measurements. This negative result of isoflavones on colonic cellular proliferation appears to be in agreement with studies conducted in other laboratories. Davies et al. (1999) reported that soy isoflavones had no inhibitory effect on the frequency of colonic tumors and Srensen et al. (1998) found that dietary soy isoflavones did not affect intestinal tumor development in APC-mutated Min mice. On the contrary, Rao et al. (1997) found that genistein at 250 µg/g diet enhanced azoxymethane-induced colon carcinogenesis in an animal model.

Another possible mechanism by which soy protein products inhibit tumorigenesis may be related to the deficiency of methionine (Hawrylewicz et al. 1995 ). The concentration of methionine in soy protein is much less than that found in casein, ~13 g/kg compared with 26 g/kg. Methionine plays a critical role in cell development because it is the precursor of S-adenosylmethine, which is the primary methyl-group donor in a large variety of methylation reactions (Cooper 1983 ). In addition, methionine is the precursor of the aminopropyl moieties of spermidine and spermine (Pegg 1988 ). It is likely that methionine could regulate cellular levels of polyamines. Although several reports have suggested that methionine and/or methionine-related metabolites might reduce the incidence of colorectal cancer (Cooper 1983 , Giovannucci et al. 1995 ), a recent study showed that a diet supplemented with methionine enhanced polyamine biosynthesis and hastened the appearance of intestinal preneoplastic changes and tumorigenesis (Duranton et al. 1999 ). However, we did not find that supplementation of 0.3% DL-methionine in the 20% soy protein diet significantly restored mucosal polyamine levels in comparison with soy protein diet alone (P < 0.06).

In conclusion, we have demonstrated for the first time that mucosal polyamine levels are altered by dietary interventions. Because the index of mucosal polyamine measurements represents colonic cancer risk, our data may offer insight into a mechanism by which dietary factors modify the risk of colorectal cancer. Consumption of soy protein could suppress mucosal polyamine levels, at least in part, through a dietary energy reduction mechanism. Further studies are warranted to support the usefulness of quantitative polyamine analysis in intestinal mucosa as an intermediate biomarker for dietary factors in experimental studies.

	ACKNOWLEDGMENTS

 
The authors thank Shoji Shibata (Department of Pharmacology, University of Hawaii) for providing rat intestinal mucosa samples in a preliminary experiment of this study.

	FOOTNOTES

 
¹ Presented in part at the 89th Annual American Association for Cancer Research Meeting, March 1998, New Orleans, LA [Wang, W. & Higuchi, C. M. (1998) Distribution of mucosal polyamine levels in rat intestinal tracts: response to dietary interventions with high- or low-risk factors. Proc. Annu. Meet. Am. Assoc. Cancer Res. 39: 16 (abs.)].

³ Abbreviations used: AcSpd, acetyl spermidine; AcSpm, acetyl spermine; Cad, cadaverine; Dad, diaminododecane; ODC, ornithine decarboxylase; Put, putrescine; SAMDC, S-adenosylmethionine decarboxylase; Spd, spermidine; Spm, spermine.

Manuscript received September 23, 1999. Initial review completed November 15, 1999. Revision accepted March 15, 2000.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Higuchi, C. M. Search for Related Content PubMed PubMed Citation Articles by , W. W. Articles by Higuchi, C. M.