Folate deficiency reduces the development of colorectal cancer in rats
Richard K. Le Leu2,
Graeme P. Young1 and
Graeme H. McIntosh
CSIRO, Health Sciences and Nutrition, PO Box 10041 Gouger Street, Adelaide BC, South Australia 5000 and
1 Department of Medicine, Flinders University, Bedford Park, South Australia 5042, Australia
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Abstract
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Alterations in folate status may play an important role in carcinogenesis. The aim of this study was to examine the effect of a diminished folate status on azoxymethane (AOM)-induced intestinal tumours in SpragueDawley rats. A total of 125 weanling male rats were divided into five equal groups and fed semi-purified diets containing either 8 mg/kg folate or no folate. After 4 weeks on experimental diets, all animals received three weekly subcutaneous injections of AOM at a dose rate of 15 mg/kg bodyweight. The animals were necropsied after 26 weeks. Rats with a diminished folate status, evident by significantly reduced blood and colonic folate concentrations and elevated plasma homocysteine levels, had significantly (P < 0.01) lower incidence and number of small intestinal and colonic tumours compared with rats displaying an adequate folate status. There was a significant decrease in the incidence of colonic adenocarcinomas (P < 0.01) and size of colonic tumours observed in the rats displaying a diminished folate status. This study shows that a diminished folate status was associated with a decrease in the development of AOM-induced colorectal cancers. The decrease in risk may be attributed to the known role of folate in cell multiplication.
Abbreviations: AOM, azoxymethane; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SCFA, short chain fatty acids.
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Introduction
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Colorectal cancer is one of the most common cancers occurring in men and women in the western world (1). Dietary factors are thought to play a predominant role in the causation of colorectal cancer. Diets high in fat and/or meat and low in fruits, cereals and/or vegetables have been found to be associated with a higher risk of colorectal cancer (2,3). The reduced risk of colorectal cancer in association with consumption of fruits, cereals and vegetables may be explained by certain micronutrients (4). A higher intake of the micronutrient folate was first proposed by Freudenheim (5) to reduce the risk of colorectal cancer. Folate deficiency is one of the most prevalent vitamin deficiencies worldwide (6). Since the casecontrol study conducted by Freudenheim (5), other epidemiological studies have tended to suggest an association between diminished folate intake and increased risk of developing colorectal cancer (7). However, none of these studies relate actual folate deficiency with increased colorectal cancer risk. Animal studies performed under controlled conditions have directly examined the relationship between folate status and colorectal carcinogenesis; however, these have been somewhat inconsistent. Studies examining the effect of folate status on the development of precancerous aberrant crypt foci (ACF) lesions suggest either a promotional effect (8) or no effect with dietary folate (9), while folate deficiency was shown to lower the number of ACF (10). Folate deficiency was found to enhance the development of colonic neoplasia induced by 1,2-dimethylhydrazine when compared with rats fed diets containing 8 mg/kg folic acid (11). In another study (12), folate deficiency increased the tumour incidence and number of tumours in rats when compared with rats consuming an adequate folate diet. In a further study, folate supplementation at 2000 mg/kg was found to have increased the colon tumour size and multiplicity in rats that had been injected with the carcinogen azoxymethane (AOM) (13), while others (9) found that folate did not affect colon tumour incidence or tumour multiplicity. Other studies have also reported conflicting results (1417). These conflicting results between these experimental animal observations may well be due to variations in experimental design, diets or to differing animal models used by researchers.
Folate is necessary for the biosynthesis of purines and thymine, as well as the maintenance of S-adenosylmethionine (SAM) levels for methylation reactions (18). An alteration in DNA methylation has been suggested to be an important factor in causing genetic instability (19,20) and is thought to contribute to carcinogenesis by affecting the expression of proto-oncogenes and/or tumour suppressor genes (21). DNA hypomethylation within the colon has been proposed as a possible mechanism by which folate deficiency might enhance colorectal carcinogenesis (22).
The purpose of this study was to determine whether a diminished folate status might alter the development of intestinal tumours induced by AOM in male SpragueDawley rats. A secondary objective was to test if DNA methylation was altered in the folate-depleted groups. Succinylsulfathiazole (a non-absorbable antibiotic drug) was employed in some dietary treatments to further enhance the level of folate depletion both before cancer initiation and during the promotional/progression phase of carcinogenesis.
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Materials and methods
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Animals and diets
A total of 125 weanling male SpragueDawley rats (70 g) were purchased from the Animal Resource Centre, Murdoch University, Perth, Australia. Animals were housed in stainless steel wire bottom cages, maintained in an air-conditioned environment 23°C ± 2°C with a 12 h light12 h dark cycle. At the age of 4 weeks they were sorted into equal bodyweights and divided into five dietary treatment groups of 25 animals. The animals were fed experimental powdered diets ad libitum and given free access to distilled water for a period of 26 weeks. Body weights were recorded weekly. After 4 weeks on experimental diets, animals were injected subcutaneously with AOM (Sigma Chemical Co., St Louis, MO) dissolved in normal saline at a dose of 15 mg/kg body weight, given weekly for 3 weeks. The rats were killed 22 weeks after the first AOM injection. The experimental diets were based on modified AIN-93 (23) semi-purified diet (Table I
). Treatment 1 (`control') contained 8 mg folate/kg diet (folate adequate). Treatment 2 (`control + S') was control with 1% succinylsulfathiazole added (Sigma Chemical Co.) for the first 4 weeks of the study. In treatment 3 (`FD') no added dietary folate was added. Rats in treatment group 4 (`FD1') consumed FD diet and 1% succinylsulfathiazole for the first 4 weeks of the study. Those in treatment group 5 (FD2) consumed FD and 1% succinylsulfathiazole for 4 weeks between weeks 8 and 12 of the study. The experimental protocol for the study is shown in Figure 1
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Fig. 1. Diagrammatic representation of experimental protocol. Control + S (`Cont + S') and FD1 contained 1% succinylsulfathiazole only for the first 4 weeks of the study, while FD2 contained 1% succinylsulfathiazole only between weeks 8 and 12 of the study.
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The experimental protocol and use of rats was approved by the CSIRO, Health Sciences and Nutrition, Animal Experimentation and Ethics Committee before commencing the study.
Sample collection and autopsy
Fresh faecal samples were collected from each rat after 4 weeks on experimental diets. Faeces of a known weight were placed in 3 ml of deionized water and homogenized; the pH was read and then the samples were stored at 20°C for SCFA measurements. The rats were killed by exsanguination while under halothane anaesthesia after 26 weeks on experimental diets. Blood was collected into EDTA-treated tubes. A portion of the blood was diluted with 1% ascorbic acid and stored at 80°C for subsequent folate analyses. The remaining blood was centrifuged at 1000 x g for 10 min and plasma was collected and stored at 80°C for subsequent homocysteine measurements. The colon and small intestine were immediately cut open longitudinally; the location, number and size of macrocopic tumours were recorded for each animal and the tumours were then removed and fixed in 10% formalin in phosphate-buffered saline for histopathological examination. Tumours were embedded in paraffin blocks, from which histological sections (4 mm) were cut and stained with haematoxylin and eosin. Tumours were identified as to type and graded with regard to malignancy and penetration according to Duke's classification (24) and classified as either adenoma or adenocarcinoma. The tumour incidence in a treatment group was defined as the percentage of rats in that group which contained at least one tumour. Tumour burden was the number of tumours per treatment group. The size and growth of the colon tumours (tumour mass index) was measured according to the following formula:
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where D1 and D2 are the diameters of the tumour.
Approximately 3 cm of fresh colon tissue (distal colon) was scraped with a glass microscope slide. The mucosal cells collected were immediately frozen in liquid nitrogen and stored at 80°C along with a piece of liver for subsequent DNA extraction and DNA methylation analysis. A section of the colon was also stored at 80°C for subsequent folate analysis.
Analytical methods
Whole blood folate was determined by HPLC with fluorescence detection (25). Total plasma homocysteine was measured by HPLC using the fluorimetric method of Vester and Rasmussen (26). Concentrations of SAM and S-adenosylhomocysteine (SAH) in the liver and colon were determined by HPLC with UV detection (27).
The DNA from colonic mucosal scrapings and a small portion of the liver was isolated using a High Pure PCR Template preparation kit (Boehringer Mannheim) followed by treatment with 10 mg/ml RNase which had been preheated to 100°C for 15 min and cooled slowly to room temperature. The DNA was subsequently purified after precipitation with ethanol. Global colonic DNA methylation status at the CpG dinucleotides was assayed using a modification of the method described by Balaghi and Wagner (28). Briefly, 2 µg of lyophilized DNA were incubated with 7.5 µCi S-adenosyl-[methyl-3H]methionine (15 Ci/mmol; Amersham) and 6 U SssI methylase (CpG) (New England Biolabs) in a final volume of 30 µl in a reaction buffer containing 10 mmol/l TrisHCl, 120 mmol/l NaCl, 10 mmol/l EDTA and 1 mmol/l dithiothreitol (DTT), pH 7.9, for 4 h at 37°C. The reactions were performed in duplicate and sampled in duplicate on to Whatman DE-81 ion exchange cellulose filters which were subsequently washed in 5% NaH2PO4 buffer for 1 h, dried, placed in a vial with 5 ml of aqueous scintillant and assayed. A higher degree of incorporation of [3H]methyl groups reflects a lower degree of DNA methylation at CpG sites.
Short-chain fatty acids in the faeces were measured by gas chromatography. Briefly, faeces were homogenized in 3 ml of water and a 200 µl aliquot was mixed with 20 µl of 0.05 mol/l 4-methyl-n-valeric acid (internal standard). The mixture was then centrifuged at 10 000 x g at room temperature for 5 min and 0.5 µl of the clear supernatant was injected directly into the gas chromatograph. Gas chromatography was performed on a Shimadzu GC-17a (Shimadzu Corporation, Kyoto, Japan) using a BPX-21 megapore capillary column (25 mx0.5 mm) (SGE, Victoria, Australia) with hydrogen flame ionization detection. Gas chromatograph conditions were as follows: injector temperature of 180°C, detector temperature of 220°C, column temperature of 110°C and helium as the carrier gas at a flow rate of 30 ml/min. A standard SCFA mixture containing acetate, butyrate and propionate was used for calculation; the results are expressed as µmol/g faeces.
Statistical analysis
Group results of biochemical markers were compared using a one-way analysis of variance test followed by Tukey's multiple comparison test. Analysis of tumour incidence was carried out using chi-squared test, and a generalized linear model with Poisson distribution of errors was used to analyse differences between treatments for tumour numbers in the large and small intestines. For tumour mass index, the data were logarithmically transformed to allow for normality of data and then analysed using a one-way analysis of variance (ANOVA). The KruskallWallis post hoc test was used after ANOVA to determine group differences. Differences between the treatment means for all tests were considered significant at P < 0.05 unless otherwise stated. Statistical analyses of tumour data were undertaken by CSIRO Mathematical and Information Services (Glen Osmond, South Australia).
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Results
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The rats in all dietary groups showed a similar weight gain; at the final week there was no significant difference among the five groups. Final body weights g (mean ± SD) were as follows: control, 535 ± 63; control + S, 554 ± 81; FD, 553 ± 74; FD1, 567 ± 59; FD2, 529 ± 48.
The incidence, number, type and size of intestinal tumours are shown in Table II
. Most of the tumours observed in all treatment groups were in the large intestine. There was no effect of the added succinylsulfathiazole treatments on tumour parameters, so treatment groups were pooled into folate-replete treatment or folate-depleted treatments. The control groups (folate-supplemented) demonstrated the highest number of tumour-bearing animals, 100% for control and 88% for control + S, whereas the folate-depleted treatments showed the lowest number of tumour bearing animals, 76% for FD, 68% for FD1 and 70% for FD2. Overall there was a significant decrease (P < 0.01) in total tumour (small intestinal + colonic) incidence and number of tumours associated with the folate-depleted treatments. Histopathological appraisal of colon tumour type showed that the animals fed the folate-depleted diets developed significantly fewer (P < 0.01) adenocarcinomas than the folate-supplemented treatment groups. There was a 71% fall in the incidence of malignant tumours in the folate-depleted treatment groups (P < 0.01).
Tumour mass index (log10) data for large intestinal tumours also showed a significant reduction (P < 0.05) in the animals maintained on folate-depleted diets, when compared with those consuming adequate folate.
Folate concentrations in the blood and colonic mucosa are shown in Table III
. Whole blood folate concentrations were significantly reduced (P < 0.001) in the treatment groups that consumed the folate-depleted diets. Colonic mucosa folate concentrations also were significantly depleted (P < 0.01) in the treatment groups that consumed the folate-depleted diets. Haematocrit values did not differ between any of the treatment groups (Table III
). Plasma homocysteine concentrations (Table III
) were significantly higher (P < 0.001) in the animals fed the folate-depleted diets. SAM concentrations in the liver (Table IV
) were significantly decreased in the folate-deficient treatments (P < 0.001). SAH concentrations in the liver were not significantly different. When expressed as a ratio (SAM:SAH) there was a significant decrease (P < 0.001) in the folate-deficient treatment groups. No significant differences were observed with SAM within the colonic mucosa for any of the treatment groups. SAH within the colonic mucosa could not be measured by HPLC due to interfering peaks.
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Table III. Haematological parameters, blood and colonic folate and plasma homocysteine values in folate-depleted and folate-replete rats
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Table IV . Hepatic and colonic SAM, SAH and hepatic and colonic methylation status in folate-depleted and folate-replete rats
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The results of DNA methylation status in the colonic mucosa are shown in Table IV
. There were no significant differences observed among the four groups in the level of unmethylated CpG within the colonic mucosa, despite the marked folate deficiency observed in the FD, FD1 and FD2 treatments.
Total SCFA concentrations in the faeces and faecal pH were significantly decreased (P < 0.01) after 4 weeks as a result of incorporation of succinylsulfathiazole at a level of 1% into the diet. SCFA concentrations (µmol/g) were as follows (mean ± SD): control, 98.1 ± 26.6; control + S, 36.3 ± 7.6; FD, 92.8 ± 27.2; FD1, 27.0 ± 7.0. pH values were as follows (mean ± SD): control, 6.9 ± 0.2; cont + S, 6.4 ± 0.1; FD, 6.9 ± 0.3; FD1, 6.2 ± 0.1.
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Discussion
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The main aim of the present study was to examine the effect of folate deficiency on intestinal tumorigenesis in the rat. The results clearly demonstrate that folate deficiency is associated with a reduction in the risk of intestinal tumorigenesis induced by AOM in rats. Total intestinal tumour incidence and the total number of tumours were significantly reduced in the folate-deficient rats relative to those consuming adequate folate. Significantly fewer adenocarcinomas were observed in the colon and rectum, and there was a reduction in the tumour mass index in the folate-deficient rats compared with the rats consuming an adequate folate diet.
Succinylsulfathiazole was added to further enhance the level of folate depletion achieved in the rats. This non-absorbable antibiotic drug inhibits the de novo synthesis of folate by the intestinal microflora of the gut. This is important in facilitating folate deficiency, as rats are known to be coprophagic, so folate produced by intestinal bacteria may be incorporated back into stores of host tissue folate (29,30). In the present study this inhibition of the intestinal microflora was evident after 4 weeks in the rats consuming succinylsulfathiazole, by significant decreases in faecal pH and in SCFA concentrations. SCFAs are produced when the intestinal microflora break down non-starch polysaccharides and resistant starch (31). In theory, by inhibiting the intestinal microflora's production of folate, a more extreme level of folate deficiency may be attained at a particular time. In a previous study (10) we demonstrated that the level of folate depletion can be enhanced in rats after feeding a folate-deficient diet supplemented with succinysulfathiazole for 4 weeks. However, the incorporation of succinylsulfathiazole into the treatments either before cancer initiation or after initiation did not further influence intestinal tumour outcomes in this study.
The reduced intestinal tumorigenesis as a result of folate deficiency in the present study was not entirely unexpected, as other experimental animal studies have also shown that folate deficiency can suppress or delay the onset of tumours (14,16,17). Folate plays an important role in nucleic acid biosynthesis and processes involved in cell proliferation (32). An association of folate supplementation with enhanced cancer induction in humans has been suggested previously (33) and may be an effect of a requirement of tumour growth for folate. Indeed, interruption of folate metabolism is a basis for anti-tumour therapy (34). In the colon, enhancement of cell proliferation is considered a risk factor for tumour development (35). Thus, a reduction in cell proliferation is a proposed mechanism for chemoprevention (36). It is plausible, therefore, that folate deficiency may well be reducing tumorigenesis by inhibiting cell proliferation. In the present study, folate deficiency supports suppression of promotion/progression events in intestinal cancer, as both the incidence and number of intestinal tumours were reduced. However, we were unable to determine whether folate is important before development of intestinal tumours; a deficiency of folate in the later stages of tumourigenesis certainly reduces tumour development. In a previous study (10) using ACF as a biomarker for colorectal cancer risk, we found no alteration in total ACF numbers in the colon between rats fed an adequate folate diet and those consuming a folate-deficient diet which results in moderate folate deficiency, suggesting that folate does not offer protection in the early stages of tumorigenesis.
The American Institute of Nutrition (AIN) diet is widely used when investigating chemopreventive agents in rat carcinogenesis studies (37,38). In the present study the background diet consisted of a semi-purified AIN-93 rodent diet that was modified to contain 12% protein supplied as casein and 20% fat supplied as a lardsunflower oil mix. In these studies, folate was fed at a level of 8 mg/kg in the folate-supplemented diets and there was no added folic acid in the folate-deficient diets. Other studies (11,12) used a specific high glutamate amino acid defined diet in showing an increased risk of colorectal cancer with folate deficiency. Folate was also at 8 mg/kg in the adequate folate diet. There was no added folic acid in the folate-deficient diet. Dimethylhydrazine was used as the carcinogen to induce colorectal carcinogenesis and the period of induction and dosage used were substantially longer (20 weeks for dosing protocol) than that of three weekly AOM injections used in the present study. It is a more artificial form of diet perturbation than was used in the present study. The contrasting results (11,12) to those of the present study are hard to explain but differences in experimental diet composition may account for differences. The larger dose for initiation than that used in the present study may offer another explanation.
Concentrations of folate in the blood and the colonic mucosa were significantly depleted in the animals fed folate-deficient diets. There was a modest rise in homocysteine only in the animals that exhibited decreased blood folate concentrations, adding further evidence of biochemically significant folate deficiency. The level of folate deficiency observed in rats in the present study can be regarded as moderate and is consistent with other studies (10,11,39) which demonstrated moderate folate deficiency. It is not necessary to resort to a pure amino acid diet to achieve such deficiency. This magnitude of folate depletion has previously been shown to be associated with a significant degree of cellular folate depletion within the colonic mucosa (40) and this has again been observed in the present study. Further evidence of moderate folate deficiency was that body growth of the animals was not affected nor was there any alteration in haematocrit or white cell levels.
Alterations in global methylation patterns are among the earliest abnormalities to occur during the development of colorectal cancer (41), although whether this is biologically significant is uncertain. Folate plays a key role in the transfer of methyl units to donor compounds (32), including many biological transmethylation reactions such as that of DNA (42). It has been proposed (11,22) that altered DNA methylation as a result of changes in folate status may be a mechanism whereby colorectal tumourigenesis is enhanced. In the present study, folate supplementation or folate deficiency did not alter global DNA methylation in the colonic mucosa. Other studies have also demonstrated that genomic DNA methylation status in the colonic mucosa is not altered by folate supplementation or folate deficiency (12,43). In the present study, colonic mucosal SAM concentrations did not change as a result of folate deficiency. SAM is the proximal methyl donor for DNA methylation reactions (42). The colon may well be resistant to changes in SAM concentrations and DNA methylation (43).
The results of the present study demonstrate that folate deficiency reduces the development of tumorigenesis right through to cancer in AOM-treated rats. This was emphasized by a reduction in intestinal tumour incidence and the smaller number of intestinal tumours observed in the folate-deficient animals. While the incidence and tumour number were not significantly reduced by folate deficiency in the large intestine, there were fewer malignant tumours and a reduction in the tumour mass index. Folate deficiency did not alter the global DNA methylation status in the colonic mucosa. It is likely that the lower tissue folate concentrations present in the folate-deficient animals may have inhibited the promotion and/or progression events of tumorigenesis.
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Notes
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2 To whom correspondence should be addressed Email: richard.leleu{at}hsn.csiro.au 
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Acknowledgments
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We thank Ben Scherer and Peter Royle for technical assistance. We would also like to acknowledge the Dairy Research and Development Corporation for their financial support of this research.
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Received May 17, 2000;
revised August 7, 2000;
accepted August 15, 2000.