Introduction: Short chain fatty acids (SCFA) are produced by large bowel fermentation of dietary carbohydrates including resistant starch (RS) and non-starch polysaccharides (NSP). SCFA (particularly butyrate) play a major role in maintaining large bowel function and may reduce the incidence of colonic disease. Butyrate is the preferred metabolic substrate of colonocytes and is believed to play a key role in modulating epithelial cell cycle, mucosal immune response and gut motility. Increasing large bowel butyrate supply requires intakes of NSP or (RS) much higher than those currently consumed in western diets. Recent studies have shown large bowel butyrate is increased by the ingestion of butyrylated starch but the characteristics and physiological effects of its ingestion in animal models of colonic disease have not yet been investigated. Aims and Methods: The experiments in in vitro and in rats described in this thesis examined: the effects of production techniques and cooking on the capacity of butyrylated starch to deliver butyrate to the large bowel. They investigated the effects of increased butyrate levels on large bowel function in: (i) normal rats, (ii) the dextran sulphate sodium (DSS) rat model of ulcerative colitis (UC) and (iii) the high dietary protein rat model of colonocyte genetic damage. Results: Starch type, pre-treatment and the degree of butyrylation influenced the in vitro digestion and fermentation characteristics of butyrylated starch before and after cooking. Butyrylated starch was less susceptible to small intestinal digestion RS as than high amylose maize starch (HAMS) in vitro. Feeding diets containing 10% cooked butyrylated starch delivered significantly greater amounts of butyrate to the large bowel of rats than 10% raw or cooked HAMS. Butyrate did not influence colonocyte proliferation throughout the large bowel of the rat but increased distal colonic IL-18 concentrations and decreased longitudinal smooth muscle contractility. Feeding HAMS or butyrylated HAMS (HAMSB) to rats during DSS induced UC and during 7 days of recovery resulted in increased mucosal damage compared to low amylose maize starch (LAMS) fed rats. When rats were fed HAMS or HAMSB during the 7 days of recovery only, there was no significant difference in mucosal damage. Genetic damage, as measured by the comet assay, was 2 fold higher in rats fed high protein diet compared with those fed a low protein diet. Concurrent feeding of high protein and either HAMS or HAMSB resulted in significantly less genetic damage. Genetic damage in rats fed 20% HAMSB was half the levels of the 20% HAMS group, and was the same as the low protein diet. Conclusions: Butyrylated starch delivered butyrate to the large bowel in rats effectively, was less susceptible to small intestinal digestion and had greater stability following cooking than the unmodified base starch. Increased digesta butyrate did not affect large bowel function or colonocyte proliferation in the normal rat; the effects on mucosal damage in the DSS rat model of ulcerative colitis were inconclusive. Increased luminal butyrate prevented high-protein induced colonocyte genetic damage. Butyrylated starches have potential to assist with the maintenance of bowel health and to contribute to reduced risk of colonic disease in the community. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1320754 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Th.--Sci. nat.--Toulouse 3, 1977. N°: 768.

We have previously shown that dietary fish oil and fiber, particularly the highly-fermentable pectin, are protective against colon cancer in a rat model of carcinogenesis. Therefore, based upon the current body of literature and our previous experimental findings, we hypothesized that one mechanism by which dietary fish oil+pectin suppress the promotion stage of colon cancer is through butyrate, the fermentation product of fiber, targeting (in particular) the p21Waf1/Cip1 gene and, via targeted histone hyperacetylation, inducing its expression. We found that dietary butyrate supplementation increased the concentration of fecal butyrate (mole %) in the distal colon, and that this increase corresponded to an increase in histone H4 acetylation. Similarly, diets supplemented with butyrate increased p21Waf1/Cip1 expression despite azoxymethane (AOM) treatment, which was not seen in non-butyrate supplemented diets. Furthermore, fish oil+butyrate diets resulted in the highest levels of apoptosis and the lowest levels of ACF, while corn oil+butyrate diets resulted in the lowest levels of apoptosis and the highest levels of ACF. Thus, it appears that the protective effect of fish oil+butyrate is due to the unique properties of fish oil, providing an environment in which butyrate’s enhancement of histone acetylation and p21 expression are pro-apoptotic, thereby diminishing pre-neoplastic ACF development.

We have previously shown that dietary fish oil and the fiber pectin protect against colon cancer in rats by increasing apoptosis induced by reactive oxygen species (ROS) at the initiation stage of tumorigenesis. We hypothesized that fish oil would incorporate into the cardiolipin of colonic mitochondrial membranes, creating an environment in which butyrate, a fermentation product of pectin, would also increase ROS and lead to apoptosis, as evidenced by decreased mitochondrial membrane potential (MMP), enhanced caspase-3 activity and cytochrome c translocation from the mitochondria, thus protecting against colon cancer by removing DNA damaged cells at the promotion stage of carcinogenesis. Sixty rats were provided a diet containing 15% corn or fish oil for 11 wk and injected with azoxymethane (AOM) or saline at wk 3 and 4. At wk 11, colonocytes were exposed to +/- butyrate ex vivo for 30 or 60 min. ROS and MMP were measured using fluorescence microscopy, and cytochrome c concentration and caspase-3 activity were measured using ELISA assays. Cardiolipin fatty acid enrichment was measured via TLC and GC. Butyrate increased ROS (p<0.0001) regardless of diet or treatment group. In colonic crypts from fish oilconsuming rats, butyrate reduced MMP (p=0.05). However, butyrate had no effect on MMP if the rats were consuming corn oil. In colonocytes from rats consuming fish oil, butyrate decreased mitochondrial cytochrome c (11%; p=0.02) concomitant with an increase in caspase-3 activity (17%; p=0.04) in the distal colon. In fish oil-fed animals, the n-3 fatty acids DHA and EPA were incorporated into cardiolipin at the expense of n-6 fatty acids. Regression analysis revealed a positive relationship between DHA (R=0.49, p=0.03) and EPA (R=0.59, p=0.02) and cytosolic cytochrome c content. As the percentage of DHA and EPA in the cardiolipin increased, the level of cytochrome c in the cytosol increased. These relationships were not seen in rats consuming corn oil and suggest that these results, induced only by the combination of butyrate with fish oil, may lead to increased apoptosis at the promotion stage of colon carcinogenesis via a mitochondria-mediated mechanism.

We have previously shown that dietary fish oil and fiber, particularly the highly-fermentable pectin, are protective against colon cancer in a rat model of carcinogenesis. Therefore, based upon the current body of literature and our previous experimental findings, we hypothesized that one mechanism by which dietary fish oil+pectin suppress the promotion stage of colon cancer is through butyrate, the fermentation product of fiber, targeting (in particular) the p21Waf1/Cip1 gene and, via targeted histone hyperacetylation, inducing its expression. We found that dietary butyrate supplementation increased the concentration of fecal butyrate (mole %) in the distal colon, and that this increase corresponded to an increase in histone H4 acetylation. Similarly, diets supplemented with butyrate increased p21Waf1/Cip1 expression despite azoxymethane (AOM) treatment, which was not seen in non-butyrate supplemented diets. Furthermore, fish oil+butyrate diets resulted in the highest levels of apoptosis and the lowest levels of ACF, while corn oil+butyrate diets resulted in the lowest levels of apoptosis and the highest levels of ACF. Thus, it appears that the protective effect of fish oil+butyrate is due to the unique properties of fish oil, providing an environment in which butyrate’s enhancement of histone acetylation and p21 expression are pro-apoptotic, thereby diminishing pre-neoplastic ACF development.

The relationship between human colonic mucosal metabolism and mucin synthesis was explored, with particular reference to ulcerative colitis (UC) and pouchitis. A hypothesis was proposed, that UC and pouchitis result from impaired metabolism of butyrate, and that the outcome of this metabolic event was reduced mucosal protection via effects on mucin synthesis. The study aims were to assess mucosal metabolism in the ileum and colon in controls and in UC, and to assess the effects of agents that are effective therapy for UC on metabolism as measured by mucin synthesis. In histologically normal colonoscopic mucosal biopsies cultured <I>in vitro</I>, the rate of metabolism of butyrate was similar in the ascending (AC) and descending colon (DC). There was a higher rate of metabolism of glutamine in the ascending colon, in agreement with previous work which stressed the relatively greater dependence of the distal colon on butyrate as an energy source. The terminal ileum (TI), in controls had a surprisingly high rate of metabolism of butyrate, significantly higher than the AC, glutamine metabolism in controls was also greater than in the AC. In ulcerative colitis (UC) the most striking change in epithelial metabolism was an increase in the rate of glutamine metabolism in the descending colon. The rates of butyrate metabolism in UC were similar to those in controls, the ratio of butyrate:glutamine metabolism was non-significantly lower in the descending colon in UC as a result of the increased rate of glutamine metabolism. Rates of metabolism in the terminal ileum were similar in UC and controls. Butyrate, at concentrations that are likely to be physiologically and pharmacologically relevant, significantly increased mucin synthesis in colonic mucosal explants from histologically normal and diseased (UC) tissue. Glucocorticoids and nicotine similarly increased colonic mucin synthesis, whereas mineralocorticoids were without effect.

Yes / Patients with adenomatous colonic polyps are at increased risk of developing further polyps suggesting field-wide alterations in cancer predisposition. The current study aimed to identify molecular alterations in the normal mucosa in the proximity of adenomatous polyps and to assess the modulating effect of butyrate, a chemopreventive compound produced by fermentation of dietary residues. A cross-sectional study was undertaken in patients with adenomatous polyps: biopsy samples were taken from the adenoma, and from macroscopically normal mucosa on the contralateral wall to the adenoma and from the mid-sigmoid colon. In normal subjects biopsies were taken from the mid-sigmoid colon. Biopsies were frozen for proteomic analysis or formalin-fixed for immunohistochemistry. Proteomic analysis was undertaken using iTRAQ workflows followed by bioinformatics analyses. A second dietary fibre intervention study arm used the same endpoints and sampling strategy at the beginning and end of a high-fibre intervention. Key findings were that keratins 8, 18 and 19 were reduced in expression level with progressive proximity to the lesion. Lesional tissue exhibited multiple K8 immunoreactive bands and overall reduced levels of keratin. Biopsies from normal subjects with low faecal butyrate also showed depressed keratin expression. Resection of the lesion and elevation of dietary fibre intake both appeared to restore keratin expression level. Changes in keratin expression associate with progression towards neoplasia, but remain modifiable risk factors. Dietary strategies may improve secondary chemoprevention. / Food Standards Agency (Ref N12017), EPSRC (EP/E036252/1), University of Sheffield

Résumé L’objectif de ce travail a été d’évaluer la toxicité du butyrate de sodium (NaBu), un inhibiteur des histones désacétylases (HDACs), et ses mécanismes d’action sur les cellules de cancer du sein humain, les cellules MCF-7 déficientes pour la caspase-3, et lignées dérivées : les cellules MCF- 7/caspase-3, et les cellules VCREMS résistantes à la vincristine, et dans une moindre mesure à la doxorubicine. La contribution de l’apoptose dans la létalité induite par le NaBu a été recherchée dans les cellules MCF-7wt en estimant l’exposition de la phosphatidylsérine ainsi que le clivage de la PARP. La présence de caspase-3, n’a ni amplifié ni accéléré l’apoptose qui a impliqué le rhéostat Bax/Bcl-2 en faveur d’une induction de Bax. La cytostasie du NaBu dans les cellules MCF-7 s’est manifestée par un blocage des cellules en phase G2/M. L’évaluation du niveau d’expression des régulateurs du cycle cellulaire dans les cellules MCF-7wt et MCF-7/caspase-3 a montré une surexpression de p21, de façon indépendante de p53. L’action cytostatique du NaBu a été associée à une accumulation légère et modeste des formes non-phosphorylées de pRB, un facteur dont la phosphorylation par les complexes cycline D/cdk4,6 et cycline E/cdk2 est nécessaire à la transition G1/S. Dans ces conditions, les niveaux de cdk2 et de Cdc25A, une oncoprotéine activatrice de cdk2, sont restés stables. Le NaBu est une molécule à effet pléïotropique, l’utilisation de la trichostatine A, inhibiteur par excellence des HDACs, a permis d’établir la relation de causalité entre l’inhibition des HDACs et la toxicité du NaBu. La plupart des inhibiteurs des HDACs induisent l’apoptose en perturbant le métabolisme oxydatif de la mitochondrie ce qui pourrait modifier le statut redox cellulaire. Nous avons cherché une implication du métabolisme du glutathion (GSH), le thiol anti-oxydant non-protéique majoritaire de la cellule, dans la toxicité induite par le NaBu. Les résultats montrent que le NaBu induit une déplétion du GSH dans les cellules MCF-7wt et dérivées de façon dose-dépendante, corrélée avec la mortalité cellulaire. Devant l’éventualité d’une consommation accrue de GSH par les enzymes associées à son métabolisme, nous avons évalué le niveau des activités des enzymes glutathion peroxydase, glutathion réductase et glutathion S-transférases. Dans les cellules MCF-7, le NaBu a induit de façon significative ces enzymes anti-oxydantes, à l’exception des GSTs, de même que la catalase, une enzyme indépendante de ce système. Les expériences visant à libérer le pool de GSH lié aux protéines ont montré que la déplétion du GSH intracellulaire est parallèle à celle du GSH lié aux protéines. Par conséquent, la consommation du GSH est réellement la cause de la chute du niveau de GSH générant un stress oxydant. La doxorubicine, un inhibiteur des topoisomérases, a une utilisation clinique limitée en raison de ses effets secondaires irréversibles (cardiotoxicité entre autres). Dans le but d’améliorer son efficacité, nous avons expérimenté des combinaisons NaBu/doxorubicine sur les cellules VCREMS et MCF-7, étant donné la capacité du NaBu à induire l’expression des topoisomérases et favoriser la conformation déployée de la chromatine. L’utilisation de la technique isobologramme nous a permis de déterminer les index de combinaison pour une application simultanée ou séquentielle des drogues. Les résultats indiquent que le NaBu sensibilise les cellules VCREMS et MCF-7 à l’action de la doxorubicine. Dans les cellules VCREMS, cet effet s’est produit en dépit de la stimulation des enzymes de détoxication, GSTs et GPX. L’ensemble de ces résultats indique que l’utilisation du NaBu en combinaison avec certains anticancéreux constitue une stratégie très intéressante en cancérothérapie.

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