Slow-release pellets of sodium butyrate do not modify azoxymethane (AOM)-induced intestinal carcinogenesis in F344 rats

Giovanna Caderni1, Cristina Luceri, Carlotta De Filippo, Maddalena Salvadori, Augusto Giannini2, Luciana Tessitore3 and Piero Dolara

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


    Abstract
 Top
 Abstract
 Introduction
 References
 
Butyrate exerts anti-tumour effects in vitro, but not consistently in vivo. We previously demonstrated that the administration of slow-release gastro-resistant pellets of sodium butyrate increases apoptosis in the colon mucosa of rats, an effect which may protect against carcinogenesis. Therefore, we studied whether the administration of butyrate pellets could protect rats against experimental colon carcinogenesis. Four to 5 week old male F344 rats were fed a high-fat (HF) diet (230 g/kg corn oil w/w) and treated s.c. with two injections (one week apart) of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight or saline. Rats were then divided into two groups: one group received sodium butyrate pellets mixed into the diet (1.5% w/w) for 33 weeks (150 mg butyrate/day) and the second group received the high-fat diet with no butyrate. Administration of sodium butyrate pellets in the diet did not significantly affect colon carcinogenesis: the number of intestinal tumours/rat was 1.6 ± 0.2 in controls and 2.1 ± 0.2 in butyrate-fed rats (means ± SE; P = 0.22, by ANOVA), while the incidence of intestinal tumours was 79 (23/29) and 90% (27/30) in controls and in butyrate-fed rats, respectively (P = 0.29 by Fisher's exact test). The level of apoptosis in the tumours was not affected by butyrate, nor was the expression of p21CIP, a cell cycle-related protein. In conclusion, the current study indicates that butyrate does not protect against AOM-induced colon carcinogenesis in rats.

Abbreviations: AOM, azoxymethane; DMH, 1,2-dimethylhydrazine; HF, high-fat; SCFA, short chain fatty acids.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Diet strongly affects the development of colon cancer, an important neoplastic disease in the Western world and it is thought that dietary intake of vegetables, and possibly fibres or starch may decrease the risk of colon cancer (1). One possible mechanism by which such dietary components may protect against cancer includes their fermentation in the colon to butyrate, since in vitro experiments have demonstrated that butyrate induces differentiation, apoptosis and increases doubling time in colon cancer cell lines (24).

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 1–2% 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 (4–5 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% sucrose–corn 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 IGo. 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 intestine—distal (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.


View this table:
[in this window]
[in a new window]
 
Table I. Frequency of intestinal tumours and mean number of adenomas or cancers/rat in AOM-induced animals treated with 150 mg butyrate/day for 33 weeks
 
The level of dysplasia in the adenomas, their dimensions and the level of cancer invasiveness were not different between the two groups (data not shown).

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 IIGo).


View this table:
[in this window]
[in a new window]
 
Table II. Apoptotic index and expression of p21CIP protein in AOM-induced rats treated with 150 mg butyrate/day for 33 weeks
 
We also measured p21CIP gene expression in histological sections of the tumours incubated in 0.3 % H2O2 for 10 min, in a microwave oven for 10 min and then with goat serum (Dako, Milan, Italy) for 20 min. Sections were then reacted with 1:100 primary mouse monoclonal antibody (Santa Cruz, CA) and with secondary antibody conjugated with the avidin–biotin peroxidase complex and stained with 3,3-diaminobenzidine and 1% hematoxylin (15,16). At least 1000 cells/sample were scored in different fields. The results relative to this determination (Table IIGo) showed that tumours in the group treated with butyrate tended to have fewer cells expressing p21CIP, although this effect was not significant.

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.


    Notes
 
1 To whom correspondence should be addressed Email: gioca{at}pharm.unifi.it Back


    Acknowledgments
 
This work was supported by EU projects FAIR CT95/0653, QLK1-1999-00346 and QLRT 1999-00505, and by Ministero Ricerca Scientifica Tecnologica (MURST), Italy. We thank Dr B.S.Reddy for helpful criticisms of the manuscript and M.Beni and P.Ceccatelli for expert technical assistance. We are also grateful to Euderma S.p.A for the preparation and kind gift of the gastro-resistant slow-release butyrate pellets.


    References
 Top
 Abstract
 Introduction
 References
 

  1. World Cancer Research Fund and American Institute for Cancer Research (1997) Food Nutrition and the prevention of cancer: a global perspective. BANTA Press, Menasha, WI, USA.
  2. Velazquez,O.C., Ledere,H.M. and Rombeau,J.L. (1996) Butyrate and the colonocyte—Implication for neoplasia. Dig. Dis. Sci., 41, 727–739.[ISI][Medline]
  3. Archer,S.Y., Meng,S., Shel,A. and Hodin,R.A. (1998) p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells. Proc. Natl Acad. Sci. USA, 95, 6791–6796.[Abstract/Free Full Text]
  4. Hague,A., Manning,A.M., Hanlon,K.A., Huschtscha,L.I., Hart,D. and Paraskeva C. (1993) Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p-53 independent pathway: implication for the possible role of dietary fiber in the prevention of large-bowel cancer. Int. J. Cancer, 55, 498–505.[ISI][Medline]
  5. Freeman,H.J. (1986) Effects of differing concentrations of sodium butyrate on 1,2-dimethylhydrazine-induced rat intestinal neoplasia. Gastroenterology, 91, 596–602.[ISI][Medline]
  6. Deschner,E.E., Ruperto,J.F., Lupton,J.R. and Newmark,H.L. (1990) Dietary butyrate (tributyrine) does not enhance AOM-induced colon tumorigenesis. Cancer Lett., 52, 79–82.[ISI][Medline]
  7. Medina,V., Afonso,J.J. Alvarez-Arguelles,H., Hernandez,C. and Gonzalez,F. (1998) Sodium butyrate inhibits carcinoma development in a 1,2-dimethylhydrazine-induced rat colon cancer. J. Parenter. Enteral. Nutr., 22, 14–17.[Abstract]
  8. Caderni,G., Luceri,C., Lancioni,L., Tessitore,L. and Dolara,P. (1998) Slow-release pellets of sodium butyrate increase apoptosis in the colon of rats treated with azoxymethane, without affecting aberrant crypt foci and colonic proliferation. Nutr. Cancer, 30, 175–181.[ISI][Medline]
  9. Samaha,H.S., Kelloff,G.J., Steele,V., Rao,C.V. and Reddy,B.S. (1997) Modulation of apoptosis by sulindac, curcumin, phenylethyl-3-methylcaffeate and 6-phenylhexyl isothiocyanate: Apoptotic index as a biomarker in colon cancer chemoprevention and promotion. Cancer Res., 57, 1301–1305.[Abstract]
  10. Reddy,B.S., Wang,C.X., Samaha,H., Lubet,R., Steele,V. and Keloff,G. (1997) Chemoprevention of colon carcinogenesis by dietary perillil alchol. Cancer Res., 57, 420–425.[Abstract]
  11. Polyak,K., Hamilton,S.R., Vogelstein,B. and Kinzler,K.W. (1996) Early alteration of cell-cycle regulated gene expression in colorectal neoplasia. Am. J. Pathol., 149, 381–387.[Abstract]
  12. European Community (1986) European Community Regulations on the Care and Use of Laboratory Animals, Law 86/609/EC.
  13. Morson.,B.C., Dawson,I.M.P., Day,D.W., Jass,J.R., Price,A.B. and Williams,G.T. (1992). Morson & Dawson's Gastrointestinal Pathology, 3rd Edn. Blackwell Scientific Publications, Oxford, pp. 608–612.
  14. Chang,W.C., Chapkin,R.S and Lupton,J.R. (1997) Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis, 18, 721–730.[Abstract]
  15. Loda,M., Cukor,B., Tam,S.W., Lavin,P., Fiorentino,M., Draetta,G.F., Jessup,J.M. and Pagano,M. (1997) Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nature Med., 3, 231–234.[ISI][Medline]
  16. Bedi,A., Pasricha,J.P., Akhtar,A., Barber,J.P., Bedi,G.C., Giardiello,F.M., Zehnbauer,B.A., Hamilton,S.R. and Jones,R.J. (1995) Inhibition of apoptosis during development of colorectal cancer. Cancer Res., 55, 1811–1816.[Abstract]
  17. Hill,M.J. (1995) Bacterial fermentation of complex carbohydrates in the human colon. Eur. J. Cancer Prev., 4, 353–358.[ISI][Medline]
  18. Watanabe,K., Reddy,B.S., Weisburger,J.H. and Kritchevsky,D. (1979) Effect of dietary alfalfa, pectin and wheat bran on azoxymethane or methylnitrosurea-induced colon carcinogenesis in F344 rats. J. Natl Cancer Inst., 63, 141–145.[ISI][Medline]
  19. McIntre,A., Gibson,P.R. and Young,G.P. (1993) Butyrate production from dietary fibre and protection against large bowel cancer in a rat model. Gut, 34, 386–391.[Abstract]
  20. Tsukamura,M., Goto,H., Arisawa,T., Hayakawa,T., Nakai,N., Murakami,T., Fujitsuka,N. and Shimomura,Y. (1998) Dietary maltitol decreases the incidence of 1,2-dimethylhydrazine-induced cecum and proximal colon tumors in rats. J. Nutr., 128, 536–540.[Abstract/Free Full Text]
  21. Young G.P., McIntyre,A., Albert,V., Folino,M., Muir,J.G. and Gibson,P.R. (1996). Wheat bran suppresses potato starch-potentiated colorectal tumorigenisis at the aberrant crypt stage in a rat model. Gastroenterology, 110, 508–514.[ISI][Medline]
  22. Sakamoto,J., Nakaji,S., Sugawara,K., Iwane,S. and Munakata,A. (1996). Comparison of resistant starch with cellulose on 1,2-dimethylhdrazine-induced colonic carcinogenesis in rats. Gastroenterology, 110, 116–120.[ISI][Medline]
  23. Cassidy,A., Bingham,S.A. and Cummings,J.H. (1994) Starch intake and colorectal cancer risk: an international comparison. Br. J. Cancer, 69, 937–942.[ISI][Medline]
  24. Burn,J., Chapman,P.D., Mathers,J., Bertario,L., Bishop,D.T., Bulow,S., Cummings,J., Phillips,R. and Vasen,H. (1995) The protocol for a European Double-blind trial of aspirin and resistant starch in familial adenomatous polyposis: the CAPP study. Eur. J. Cancer, 31A, 1385–1386.
  25. D'Argenio,G., Cosenza,V., Delle Cave,M., Iovino,P., Della Valle,N., Lombardi,G. and Mazzacca,G. (1996). Butyrate enemas in experimental colitis and protection against large bowel cancer in a rat model. Gastroenterology, 110, 1727–1734.[ISI][Medline]
  26. Wang,J. and Friedman,E.A. (1998) Short-chain fatty acids induce cell cycle inhibitors in colonocytes. Gastroenterology, 114, 940–946.[ISI][Medline]
  27. Lupton,J.R. and Turner,N.D. (1999) Potential protective mechanisms of wheat bran fiber. Am. J. Med., 106, 24S–27S.[Medline]
Received September 28, 2000; revised September 28, 2000; accepted December 5, 2000.