The Journal of Nutrition Vol. 128 No. 5 May 1998, pp. 804-809

A Diet Containing Chickpeas and Wheat Offers Less Protection against Colon Tumors than a Casein and Wheat Diet in Dimethylhydrazine-Treated Rats^1,2

Graeme H. McIntosh³, Y. H. Alex Wang⁴, and Peter J. Royle

CSIRO Division of Human Nutrition, Adelaide, South Australia 5000

	ABSTRACT

Abstract Introduction Methods Results Discussion References

We examined the influence of extruded chickpeas and wheat relative to casein and wheat in a dimethylhydrazine (DMH)-induced colon tumor study in male Sprague-Dawley rats. The three diets, based on a modified AIN76 rodent diet with fat present at 10 g/100 g dry matter (DM), were as follows: casein with wheat starch (Cas/S) as control, casein with wheat (Cas/W) and chickpeas with wheat (CP/W). All diets were fed from 5 wk of age throughout the 28-wk study. At 28 wk, there was a significantly lower incidence of large intestinal tumors in rats fed Cas/W relative to those fed CP/W ( 11 vs. 56%, chi-square test, P = 0.018). The colonic tumor burden (tumors/tumor-bearing animal) was not different in Cas/W-fed and CP/W-fed rats (1 vs. 1.7), but the tumor mass index was significantly lower in the former group (0.22 vs. 1.21, P = 0.026). Rats fed the CP/W diet had significantly lower plasma cholesterol concentration (P < 0.01) than rats fed the other two diets. The cecal contents of rats fed the CP/W diet had significantly greater relative weights (46%, P < 0.05) than those of the Cas/W-fed rats; this was associated with higher concentrations of all short-chain fatty acids. Fecal analyses showed significantly (P < 0.05) higher concentrations of total fat (54%), total steroids (83%) and secondary bile acids (179%) in the CP/W-fed rats relative those fed Cas/W. There were higher concentrations of nitrogen in the feces of CP/W rats relative to the Cas/W-fed rats (84%, P < 0.05), associated with greater fecal weights (67%, P < 0.05). Although wheat and its fibers have been shown to be protective against DMH-induced cancers in rats, this was not the case in this study in which chickpeas (45 g/100 g diet) provided the protein and were an important source of soluble fiber. Elevated fat, secondary bile acid concentrations and/or nitrogenous compounds could be responsible for the increased colon tumorigenesis seen and may reflect a legume effect.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The potential for dietary ingredients to promote or inhibit the expression of colon cancer in communities is being examined via population and experimental studies to help define a more protective diet (Riboli and Cummings 1993). There is good evidence that whole grains (wheat in particular) are effective in cancer prevention (Freudenheim et al. 1990, Jacobs et al. 1995, Jensen et al. 1982, Rosen et al. 1988, Trock et al. 1990). Another important source of dietary fiber, legumes, have received less attention, and the evidence concerning their effect is of a negative or nil influence (Bauer et al. 1981, Haenszel et al. 1973, Jacobs and Lupton 1986). Most legumes are a good source of dietary fiber; the major component () of cotyledonary fiber is soluble, consisting of pectic polysaccharides. There are also cellulose (insoluble) and hemicellulose (such as arabinoxylan) fiber components (Sgarbieri, 1989). Oligosaccharides and resistant starch (high amylose) should also be considered as part of the fiber component because they too are not digested in the small intestine (Muir et al. 1994a, Rumney and Rowland 1995). They are well represented in these legumes and contribute to a lower digestibility coefficient, as well as having a bulking effect in the large intestine, where they are subject to fermentation.

One experimental model used to evaluate diet strategies has been the rodent 1,2-dimethylhydrazine dihydrochloride (DMH)⁵ colon cancer model (Goldin 1988, Pozharisski 1973), which is useful because of the similar pathology between this model and the human disease. Although tumors in the small intestine also develop with this cancer model, they appear to be in relatively constant proportion to large intestinal tumor expression and are present in all rodent models of this disease. With this animal model, an examination of the influence of dietary fiber and protein sources has been undertaken (McIntosh et al. 1995); cereal fiber sources such as wheat and barley bran have also been found to be protective, relative to other fiber and/or bran types (McIntosh et al. 1996).

Because some epidemiologic reports have indicated that legumes fail to protect from colon cancer (Potter 1996), this study examines the influence of a legume (chickpea, Cicer arietinum) on the risk of colon cancer. The chickpea has been compared with casein as a protein source for its influence on colon cancer (when fed with whole wheat) in the rat DMH colon cancer model. Chickpeas are a good source of protein as well as energy. To remove antinutrients that might otherwise significantly influence outcome, wheat and chickpeas were extruded (heat, moisture and pressure treatment); in a previous study (Wang and McIntosh 1996), this was shown to remove enzyme inhibitors and significantly improve growth in young rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Male Sprague-Dawley rats from the CSIRO Division of Human Nutrition animal colony were used. They were housed in suspended stainless steel wire cages in a clean air-conditioned environment (23 ± 2°C), with a 12-h light:dark cycle. Rats (18 per treatment) were introduced to the dietary treatments at 5 wk of age and were fed these diets for the next 28 wk. Rats consumed food and water ad libitum.

Diets were based on the AIN76 formula (AIN 1977) with the following food ingredients used as sources of protein: casein [85g protein/100 g dry matter (DM)] and wheat (9.4 g/100 g DM) were used in the second diet (Cas/W) and chickpeas (28.2 g/100 g DM) and wheat in the third diet (CP/W); in the control diet (Cas/S), casein was used with purified wheat starch (34.6 g/100 g DM) (Table 1). Casein was manufactured and supplied by Bonlac Foods, Melbourne, Australia. Extruded chickpeas (desi type) and wheat (Janz cultivar, central NSW, Australia) were supplied by Allgold Foods (Leeton NSW, Australia).

Wheat starch was supplied by Goodman Fielder, Sydney, Australia; sucrose by Colonial Sugar Refineries, Sydney, Australia; sunflower seed oil (SSO) by Nuttelex, Melbourne, Australia; and lard from George Chapman, Seaton, Australia. Cellulose was supplied as -cellulose (Sigma Chemical, Sydney, Australia). Protein was provided at a final concentration of 5 g/100 g. Total fat was supplied at 10 g/100 g in the diet by adding a mixture of SSO/lard (1:1) including fat content of the other dietary components. Other major dietary components such as carbohydrate or fiber were also adjusted to provide diets of comparable composition (Table 1). Dietary fiber and starch concentrations for extruded chickpeas were 8.3 and 46.3 g/100 g DM, respectively, and for extruded wheat were 10.1 and 66.9 g/100 g DM, respectively. The diets were mixed, pelleted, dried to a constant moisture level of 8-10% at 35°C and stored at 0-4°C until used. The diets were prepared every 4-6 wk to minimize loss of palatability or loss of nutrient value. Analytical checks were made periodically by using vitamin E (-tocopherol) and calcium as markers to confirm that the diets met specifications.

Carcinogen and necropsy methodology.  At 9 wk of age, the rats were injected with the procarcinogen DMH (Aldrich Chemical, Milwaukee, WI) suspended in an isotonic saline solution (pH ~7). Three subcutaneous injections in the inguinal region were given at weekly intervals at a dose of 15 mg/kg body weight. After each injection, the rats were kept in cages in a fume hood for 48 h to meet safety requirements with regard to the use of the procarcinogen. The basic methodology for this approach has been published previously (McIntosh et al. 1995 and 1996). Rats were weighed and placed in metabolism cages at 24 wk of age to enable daily food intakes, and fecal and urine outputs to be measured.

Rats were weighed weekly and observed daily for any untoward signs, particularly during the last 4-6 wk of experimentation. Symptoms indicative of active tumors were regularly monitored and included the following: loss of body weight, fecal blood, ruffled fur and anemia. When such symptoms were diagnosed (usually at 20-24 wk after DMH treatments), the rats were killed and the experiment terminated. The majority of tumors, however, were identified readily at necropsy by gross examination of the walls of the small and large intestine.

All procedures used in this study were reviewed and approved by the Animal Experimentation Ethics Committee (CSIRO Division of Human Nutrition) before commencement and met the principles of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (Australian Government Publishing Services, Canberra, Australia).

Rats were anesthetized with ether and killed by bleeding from the abdominal aorta. The entire gastrointestinal tract was removed, opened and the number, size and location of intestinal tumors carefully recorded. Samples of liver, normal colon, tumors, kidney and spleen were taken and stored at 80°C for biochemical analysis. If identification of tumors was not possible by gross examination, and to characterize tumor type (adenocarcinoma or adenoma), tissues were fixed in formol saline and subsequently assessed histologically. The majority of tumors, however, were identified readily at necropsy by close examination. Cecal contents were collected and stored in 9 g/L saline and frozen at 20°C until short-chain fatty acid (SCFA) analysis was undertaken, within 1 mo.

Blood samples were collected into EDTA containers at termination of the experiment and were analyzed for hematocrit; the plasma was removed and stored frozen (20°C) for subsequent analyses.

Chemical and biochemical analyses.  Nitrogen of legumes and feeds was analyzed in finely ground and dried samples by using an NA1500 Nitrogen Analyzer (Carlo Erba, Milan, Italy). Total dietary fiber and starch of extruded chickpeas and wheat were determined according to AOAC methods (1990). Content of trypsin inhibitors in extruded chickpeas and wheat was analyzed by using the method described by Saini et al. (1993) and were found to be negligible. The lipid content of legumes was measured by the method of Behling et al. (1990) and involved total lipid extraction with chloroform/methanol according to the method of Folch et al. (1957), modified to include the extraction of acid-soluble lipid material. Short-chain fatty acids were assayed by the method of Whitehead et al. (1976). Briefly, an aliquot of a thawed and homogenized sample (600 µL for feces, 300 µL for cecal contents) was added to 300 µL ether, 60 µL concentrated H₂SO₄, 20 µL methyl valerate (Sigma Chemical, St. Louis, MO) as the internal standard and 0.35 g NaCl in a capped tube. After gentle mixing for 2 min and centrifugation at 10,000 × g for 5 min, a 5-µL aliquot of the top ether phase was injected into a gas chromatograph (Perkin Elmer 3920, Norwalk, CT, column temperature 170°C). A standard SCFA mixture containing acetate, butyrate and propionate was used for calculations of the SCFA concentrations and the results were expressed as micromoles per gram of wet feces. Calcium, magnesium and vitamin E used for assessment of diet composition, feces and urine were analyzed by atomic absorption spectroscopy (Varian Spectra AA, Melbourne, Australia) and HPLC (McIntosh et al. 1988). Neutral steroids and bile acids in feces were analyzed by gas liquid chromatography (Glatz et al. 1985). Nitrogen was analyzed by an NA 1500 nitrogen analyzer (Carlo Erba).

Statistical methods.  Tumor incidence was reported as the number of rats per group showing evidence of tumors, tumor burden as the total number of tumors in each dietary group, and tumor mass index (TMI) was calculated according to the following function: TMI = log₁₀(S₁ⁿ[D₁ + D₂/2]² + C) where D₁ and D₂ are the diameters of the tumor and C is a constant (1.57) added to the total area to avoid the calculation log₁₀ of zero (McIntosh et al. 1996). Tumor incidence between treatment groups was compared using the chi-square test. Other statistical calculations were performed using a one-way ANOVA. Differences between the means were then subjected to the Kruskall-Wallis or Tukey-Kramer multiple comparison test (Steel and Torrie 1980) with significance expressed at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

There were no differences in body weights of rats at termination (28 wk after commencing diets, Table 2). There was a significantly lower plasma cholesterol concentration (18%, P < 0.01) in the CP/W-fed rats relative to those fed Cas/S or Cas/W. (Fig. 1). Rats fed the casein-containing diets did not differ. There was a significantly greater (~43%) cecum weight in rats fed the CP/W diet than in rats fed Cas/S and Cas/W, which did not differ (Table 2). There were no differences in colon weights among the three groups. The relative weights of kidneys from CP/W-fed rats were less than those of rats fed Cas/W and Cas/S (12 and 16% P < 0.05), which did not differ from one other.

View larger version (25K): [in this window] [in a new window]   Fig 1. Plasma cholesterol concentration of rats after consuming diets containing casein and wheat starch (Cas/S, control), casein and wheat (Cas/W) or chickpea and wheat (CP/W) for 28 wk. Values are means ± SD, n = 18. Bars with different letters are significantly different, P < 0.01 (Tukey-Kramer).

Total tumor data were not significantly different between treatments (Table 3). The large intestinal tumor incidence, however, was significantly lower (P = 0.018) for rats fed Cas/W relative to those fed CP/W, but the tumors per tumor-bearing animal (burden) did not differ. The TMI differed significantly between these two treatment groups (P = 0.026). There were no significant differences between tumor data means for rats fed Cas/S and Cas/W. A constant ratio of adenomas to adenocarcinomas (1:1) occurred in all diet treatment groups.

Butyrate, propionate and acetate concentrations were significantly higher in cecal contents of rats fed CP/W than in the Cas/W-fed group (Table 4) (38, 53 and 37%, respectively). Cecal concentrations of butyrate and propionate were significantly lower in Cas/S-fed rats relative to Cas/W-fed rats (51 and 39%, respectively), but acetate concentration did not differ.

Fecal output weight was significantly greater in rats fed the CP/W diet relative to those fed the Cas/S or Cas/W diets (P = 0.047) (Table 5). Fecal nitrogen concentration was significantly higher (P < 0.001) in rats fed CP/W relative to those fed Cas/W and Cas/S, which did not differ. Fecal magnesium concentration was 64% greater (P < 0.05) in rats fed CP/W (8.24 ± 0.37 mg/g DM,) than in those fed Cas/W (5.03 ± 0.43) and in those fed Cas/W than in the Cas/S-fed rats (2.71 ± 0.27) (P < 0.05).

Concentrations of total fat, total steroids and secondary bile acids were significantly higher (P < 0.05) in the feces of CP/W-fed rats compared with those fed Cas/W (54, 83 and 179%, respectively) (Table 6). Values for Cas/W-fed rats did not differ significantly from those of Cas/S-fed rats.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In this experiment, the chickpea/wheat diet sustained growth, lowered the concentration of circulating plasma cholesterol and significantly increased the fermentative activity and mass in the ceca of rats compared with rats fed Cas/W. However, these differences were not reflected in the relative colon weights. They were associated with a significantly greater incidence of tumors in the large intestines of rats fed CP/W diet relative to rats fed Cas/W. This result suggests that the addition of chickpea is responsible for this change. Wheat is generally considered to be protective against colon cancer (Jacobs et al. 1995), and limited data in this study (Cas/W vs. Cas/S) reflected such a trend, but were not significant (P = 0.075).

More than half of dietary fibers contributed by chickpeas are soluble, which lowered plasma cholesterol concentration significantly and was associated with significantly increased fermentative activity in the cecum, as shown by SCFA production and secondary bile acid concentrations. Resistant starch is also present in extruded chickpeas and wheat and may have contributed to this effect, although it was not assayed in these ingredients. The fibers from chickpeas and wheat could be making an important soluble fiber-like contribution. Increased luminal cholesterol and bile acids may increase the risk of toxic metabolites being formed. The cytotoxic nature of secondary bile acids and free fatty acids (FFA) would have a promotional influence on colon carcinogenesis (Nair 1988, Govers et al. 1993). In this respect, Govers et al. reported a soybean protein effect, relative to casein in the colon of rats, of increased FFA, cell cytotoxicity, cell proliferation and fecal alkaline phosphatase. Although we did not measure FFA in this study, the greater concentrations of secondary bile acids in rats fed the CP/W diet could have had an influence on large bowel tumor development. The level of fat (10 g/100 g) used in our experiment should allow the expression of a protein influence on bile acid concentration, an effect that is not seen when fat in the diet is 20 g/100 g (Govers et al. 1993).

McIntyre et al. (1993) proposed that rapid early fermentation in cecal and proximal colon is not consistent with protection of the distal colon because of the greatly diminished concentrations of butyrate past the proximal colon. Butyrate is recognized as a differentiating and antineoplastic agent from in vivo and in vitro studies with neoplastic colonic cells (Lupton 1995). However, as Lupton points out, the influence of SCFA (and butyrate in particular) could be very different in neoplastic cells compared with normal colonocytes, and increased proliferation could be the response to elevated concentrations of butyrate in normal colonocytes. Whether this would lead to increased rather than decreased risk is a matter of ongoing speculation (Johnson 1995, Lupton 1995, Wasan and Goodlad 1996). The increased tumorigenesis associated with soluble dietary fiber supplements (e.g., guar gum, pectin or oat bran) in the DMH colon cancer model (Bauer et al. 1981, Jacobs and Lupton 1986, Jacobs 1987) has supported the above hypothesis. However, there may be a hierarchy of influences, with the increased concentrations of secondary bile acids associated with diets rich in soluble fiber being dominant in this respect. Under other conditions, increased SCFA production could be dominant and protective.

There is considerable interest in the potential of oligosaccharides and resistant starch to provide protection against cancer (Cassidy et al. 1994, Howard et al. 1995, Rumney and Rowland 1995, Van Munster et al. 1994); their benefit is attributed to an increase of "desirable" fermentative bacteria in the large intestines and their by-products. Insofar as extruded chickpeas contain 5-15 g/100 g DM as resistant starch (Muir et al. 1994b) and ~2.8 g/100 g DM oligosaccharides (G. McIntosh, unpublished data), the present data do not support such an effect. However, the addition of chickpeas also resulted in an increased excretion of secondary bile acids, which, as noted earlier, are themselves a recognized risk factor for colon cancer.

Another component of the cecal contents are the nitrogenous compounds, which presumably include undigested proteins; these may be broken down to potentially toxic metabolites. The fecal nitrogen output in the CP/W-fed rats was more than double that of the Cas/W-fed rats, and this was associated with significantly lower relative kidney weights. This increased fecal nitrogen could be due in part to the diversion of endogenous nitrogen (urea) into the cecum/colon, as has been reported previously with legume ingestion (De Oliveira and Sgarbieri 1986), with supplemented oligosaccharides (Younes et al. 1995) and with dietary fiber generally (Gallaher and Schneeman 1992). Of particular interest is the type of end products of fermentation. Nitrosation compounds may result from such microbial activity and some of these (e.g., nitrosamines) have been associated with increased cancer risk (Bingham et al. 1996, Hill 1996). Others have proposed that a high ammonia output could result in substantial damage to the colon wall, leading to increased proliferation and risk (Visek and Clinton 1991). Rumney and Rowland (1995), working with human fecal flora-infected rats, found that feeding soybean oligosaccharides decreased the output of nitrosation compounds. Some of the increased nitrogen will undoubtedly be as microbial protein. Clearly there is a need for further elucidation of the nature of these influences on colonic metabolism and their effect on colon cancer risk. Fecal magnesium has also been used as a marker of increased colon cell proliferation and cancer risk in the large bowel (Govers et al. 1993). In this study increased tumor risk was not consistent with increased fecal magnesium excretion.

Although no single or specific factor has been identified to account for the higher incidence of tumors in DMH-treated rats fed chickpeas compared with those fed casein, it is an observation that deserves further careful investigation. The study does establish, however, that although wheat might provide protection under certain specified conditions, these benefits may be abolished when wheat is combined with a grain legume such as chickpeas, as was the case in this study. Clearly, the confusion that surrounds dietary fibers and their influence on cancer risk requires further investigation.

	ACKNOWLEDGMENTS

We wish to acknowledge the assistance of Ben Scherer, Richard Le Leu and Sally Elieff with this work. Angela Reid, CSIRO Biometrics Unit in Adelaide, assisted with some of the statistical assessment.

	FOOTNOTES

Manuscript received 31 July 1997. Initial reviews completed 5 September 1997. Revision accepted 3 December 1997.

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