Department of Pharmacology of the University of Florence, Viale G. Pieraccini, 6, 50139, Florence,
2 Department of Pathology, S.M. Annunziata Hospital, Florence and
3 Dipartimento di Scienze Chimiche Alimentari Farmaceutiche e Farmacologiche, Università del Piemonte Orientale `Amedeo Avogadro', Italy
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Abstract |
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Abbreviations: AOM, azoxymethane; DMH, 1,2-dimethylhydrazine; HF, high-fat; SCFA, short chain fatty acids.
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Introduction |
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However, carcinogenesis experiments in which butyrate has been administered directly in vivo have produced conflicting results (57). Freeman (5), for example, demonstrated that the administration of 12% sodium butyrate in drinking water to 1,2-dimethylhydrazine (DMH)-treated rats significantly increased the number of colonic tumours. In another study, 5% glycerol tributyrate added to the diet did not affect tumourigenesis in the colon of mice treated with azoxymethane (AOM) (6) and in a third trial, the intracaecal administration of sodium butyrate during the initiation phase of colon carcinogenesis by DMH significantly reduced the development of colon cancer in rats (7).
We recently demonstrated (8) that 1.5% sodium butyrate, given as gastro-resistant slow-release pellets, did not affect AOM-induced preneoplastic lesions or proliferative activity in rats, but increased apoptosis in the colonic mucosa, an effect which may protect against colon carcinogenesis (9,10). On this basis, we decided to investigate whether butyrate, administered as gastro-resistant slow-release pellets to rats, would affect AOM-induced colon carcinogenesis. We also studied the level of apoptosis in the colonic tumours and the level of expression of the p21CIP gene, the prototype inhibitor of cyclin-dependent kinases implicated in colorectal carcinogenesis (11) and regulated in vitro by butyrate (3).
The experiment was carried out on male F344 rats (45 weeks old) (Nossan, Correzzana, Milan, Italy) according to the European Union Regulations on the Care and Use of Laboratory Animals (12). Gastro-resistant slow-release pellets [containing 66 g sodium butyrate (Merck, Italy)/100 g pellets] were prepared by Euderma (Rimini, Italy) (8). This pharmaceutical preparation reaches the colon, with a peak in luminal concentration in the caecum at 16 h. Rats fed with these pellets had a significant increase in the concentration of butyrate in the faeces (8). Animals (n = 99) were fed a high-fat (HF) diet whose composition is based on the AIN76 diet, modified to contain a high amount of fat (230 g/kg corn oil w/w), a low level of cellulose (20 g/kg w/w) and a low level of calcium (1.3 g/kg w/w) (8). Rats were treated s.c. with two injections (1 week apart) of AOM (Sigma, Italy) (15 mg/kg, total dose 30 mg/kg); control rats received an equal volume of normal saline. One week after the last injection with AOM, both controls and carcinogen-treated rats were divided into two groups and received one of the following diets: butyrate pellets mixed into the diet to give a final concentration of 1.5% (w/w) butyrate or the same pellets containing a mixture of 75:25% sucrosecorn starch (placebo pellets). We therefore had four groups: groups A (n = 14) and B (n = 15) were controls treated with saline and administered placebo or butyrate pellets, respectively; and groups C (n = 30) and D (n = 30) were treated with AOM and administered placebo or butyrate pellets, respectively. The weight of the animals at the end of the experiment (33 weeks after the first AOM injection) was similar among the different groups [480 ± 7 (13); 484 ± 6 (15); 458 ± 7 (29) and 462 ± 6 (30), in groups A, B, C and D, respectively; means (g) ± SE (number of rats/group)]. Two rats in the groups A and C died before the end of the experiment. No apparent pathological findings were found during autopsy of these animals, that were excluded from the valuable number of each group.
At autopsy, no tumours were found in the control rats treated with saline (groups A and B). The number of adenomas or cancers/rat and the frequency of intestinal tumours (both adenomas and cancers) in the AOM-treated animals are shown in Table I. While the number of adenomas was the same in the two groups, the number of cancers in the butyrate group was higher than in rats treated with placebo pellets, although this difference was not significant (P = 0.10). Similarly, the frequency of intestinal tumours (number of rats with tumours/rats in the group) was slightly higher in butyrate-treated rats than in controls although the difference was not significant (P = 0.29). The distribution of tumours in the different subsites of the intestinedistal (towards the anus), mid and proximal (the herringbone part of the colon plus the caecum)was similar (data not shown). No tumours were found in the small intestine of butyrate-treated rats.
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Since it has been suggested that an increase in apoptosis may account for the chemopreventive effect of several agents in colon carcinogenesis (9,10,14), we also studied the level of apoptosis in paraffin-embedded sections stained with hematoxylin-eosin, according to Samaha et al. (9) and Reddy et al. (10). At least 1000 cells/sample were scored. The results of this determination showed no difference between the two groups (Table II).
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The results of this study show that sodium butyrate, administered as gastro-resistant slow-release pellets, does not significantly affect AOM-induced intestinal carcinogenesis, apoptosis or p21CIP gene expression in the colonic mucosa. Indeed, considering the results relative to the number of cancers/rat, our study might even suggest a stimulating effect of butyrate on colon carcinogenesis.
The hypothesis that butyrate may inhibit colon carcinogenesis is based on results obtained with a variety of cell lines in vitro, including colonocytes, in which butyrate inhibits the growth of neoplastic cells, induces their differentiation and enhances apoptosis (24). Another line of evidence supporting the `butyrate hypothesis' derives from epidemiological and experimental studies showing that diets rich in complex carbohydrates (especially fibres, but also starch) are associated with a lower risk of colon cancer (1). In mammalian intestine such foods are fermented into short chain fatty acids (SCFA), mainly acetate, propionate and butyrate, at a molar ratio of 60:25:15 (2), a proportion that may vary according to the complex carbohydrates and to the bacteria involved in the fermentation (17). Fibres such as wheat bran, or maltitol which increase butyrate, especially in the distal part of the colon, have been reported to protect against experimental carcinogenesis (1820), although other studies with different butyrate-producing foods have not confirmed this protective effect (21,22). It has also been suggested that the negative correlation between colon cancer and starch intake in different countries may be due to the high production of butyrate from starch fermentation (23). Accordingly, an intervention trial with resistant starch is being carried out, with the hope that increased intestinal butyrate may decrease colon cancer in high risk subjects such as familial adenomatous polyposis patients (24).
However, previous studies and the present one, in which sodium butyrate was administered directly to animals, have not confirmed these optimistic possibilities (5). It has been suggested (6), that the adverse effect of sodium butyrate on colon carcinogenesis (5) might be due to sodium. This does not seem to be the case since in a recent study by Medina et al. (7); sodium butyrate, administered directly into the caecum of rats during DMH-induction, reduced colon carcinogenesis. Similarly, the higher susceptibility to AOM-induced cancer observed in rats with experimental colitis is lowered by sodium butyrate enemas (25). At variance with previous studies (7,25), in which butyrate was administered directly into the caecum or rectum, we used slow-release gastro-resistant pellets mixed into the diet. We though that a similar pharmaceutical preparation, if active, could even be used in humans at high risk, in which direct administration of butyrate by enema is problematic. The preparation we used reaches the colon, and although it does not modify colonic proliferation or AOM-induced aberrant crypt foci, it increases apoptosis in the distal colon (8). Notwithstanding these promising effects, the butyrate pellets in our study did not modify colon carcinogenesis.
It is possible that a more elevated level of luminal butyrate is necessary to affect carcinogenesis. One possible alternative could be that the real link between complex carbohydrates and cancer is the pattern of fermentation products formed by these foods, and not only butyrate. Accordingly, it has been reported (26) that the induction of p21CIP, a cell cycle inhibitor, is lower when cells are exposed to butyrate alone than to a combination of SCFA, mimicking the molar ratio produced by the fermentation of wheat bran in the colon. It has also been demonstrated that although oat bran produces a higher amount of butyrate by fermentation than wheat bran, it is less active in reducing colon carcinogenesis (27). This suggests that butyrate is not the only link between fibre fermentation and cancer.
In conclusion, our results clearly show that butyrate administered in the form of gastro-resistant pellets does not protect against experimentally induced colon carcinogenesis in rats. Further carcinogenesis studies involving direct administration of SCFA solutions containing different proportions of butyrate or the direct production of butyrate by fermentation into the various colon subsites are probably necessary for a better understanding of the role of SCFA and butyrate on colon cancer development.
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Notes |
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Acknowledgments |
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References |
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