Effect of colonic bacterial metabolites on Caco-2 cell paracellular permeability in vitro.

One common effect of tumor promoters is increased tight junction (TJ) permeability. TJs are responsible for paracellular permeability and integrity of the barrier function. Occludin is one of the main proteins responsible for TJ structure. This study tested the effects of physiological levels of phenol, ammonia, primary bile acids (cholic acid, CA, and chenodeoxycholic acid, CDCA), and secondary bile acids (lithocholic acid, LCA, and deoxycholic acid, DCA) on paracellular permeability using a Caco-2 cell model. Paracellular permeability of Caco-2 monolayers was assessed by transepithelial electrical resistance (TER) and the apical to basolateral flux of [14C]-mannitol. Secondary, but not primary, bile acids increased permeability as reflected by significantly decreased TER and increased mannitol flux. Both phenol and ammonia also increased permeability. The primary bile acid CA significantly increased occludin expression (P < 0.05), whereas CDCA had no significant effect on occludin expression as compared to the negative control. The secondary bile acids DCA and LCA significantly increased occludin expression (P < 0.05), whereas phenol had no significant effect on the protein expression as compared to the negative control. This suggests that the increased permeability observed with LCA, DCA, phenol, and ammonia was not related to an effect on occludin expression. In conclusion, phenol, ammonia, and secondary bile acids were shown to increase paracellular permeability and reduce epithelial barrier function at doses typical of levels found in fecal samples. The results contribute to the evidence these gut microflora-generated products have tumor-promoting activity.

Fecal water as a non-invasive biomarker in nutritional intervention: comparison of preparation methods and refinement of different endpoints.

The assessment of cellular effects by the aqueous phase of human feces (fecal water, FW) is a useful biomarker approach to study cancer risks and protective activities of food. In order to refine and develop the biomarker, different protocols of preparing FW were compared. Fecal waters were prepared by 3 methods: (A) direct centrifugation; (B) extraction of feces in PBS before centrifugation; and (C) centrifugation of lyophilized and reconstituted feces. Genotoxicity was determined in colon cells using the Comet assay. Selected samples were investigated for additional parameters related to carcinogenesis. Two of 7 FWs obtained by methods A and B were similarly genotoxic. Method B, however, yielded higher volumes of FW, allowing sterile filtration for long-term culture experiments. Four of 7 samples were non-genotoxic when prepared according to all 3 methods. FW from lyophilized feces and from fresh samples were equally genotoxic. FWs modulated cytotoxicity, paracellular permeability, and invasion, independent of their genotoxicity. All 3 methods of FW preparation can be used to assess genotoxicity. The higher volumes of FW obtained by preparation method B greatly enhance the perspectives of measuring different types of biological parameters and using these to disclose activities related to cancer development.

Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients.

BACKGROUND: Animal studies suggest that prebiotics and probiotics exert protective effects against tumor development in the colon, but human data supporting this suggestion are weak. OBJECTIVE: The objective was to verify whether the prebiotic concept (selective interaction with colonic flora of nondigested carbohydrates) as induced by a synbiotic preparation-oligofructose-enriched inulin (SYN1) + Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (BB12)-is able to reduce the risk of colon cancer in humans. DESIGN: The 12-wk randomized, double-blind, placebo-controlled trial of a synbiotic food composed of the prebiotic SYN1 and probiotics LGG and BB12 was conducted in 37 colon cancer patients and 43 polypectomized patients. Fecal and blood samples were obtained before, during, and after the intervention, and colorectal biopsy samples were obtained before and after the intervention. The effect of synbiotic consumption on a battery of intermediate bio-markers for colon cancer was examined. RESULTS: Synbiotic intervention resulted in significant changes in fecal flora: Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased. The intervention significantly reduced colorectal proliferation and the capacity of fecal water to induce necrosis in colonic cells and improve epithelial barrier function in polypectomized patients. Genotoxicity assays of colonic biopsy samples indicated a decreased exposure to genotoxins in polypectomized patients at the end of the intervention period. Synbiotic consumption prevented an increased secretion of interleukin 2 by peripheral blood mononuclear cells in the polypectomized patients and increased the production of interferon gamma in the cancer patients. CONCLUSIONS: Several colorectal cancer biomarkers can be altered favorably by synbiotic intervention.

The potential mechanisms involved in the anti-carcinogenic action of probiotics.

Probiotic bacteria are live microbial food ingredients that provide a health benefit to the consumer. In the past it was suggested that they served to benefit the host primarily through the prevention of intestinal infections. More recent studies have implicated probiotic bacteria in a number of other beneficial effects within the host including: *The suppression of allergies. *Control of blood cholesterol levels. *Modulation of immune function. *And the prevention of cancers of the colon. The reputed anti-carcinogenic effect of probiotics arises from in vivo studies in both animals and to a limited extent in man; this evidence is supported by in vitro studies with carcinoma cell lines and anti-mutagenicity assays. However, the mechanisms involved in any effect have thus far been difficult to elucidate; studies offer evidence for a variety of mechanisms; we have reviewed these and come to the opinion that, the anti-carcinogenic effect may not be attributable to a single mechanism but rather to a combination of events not yet fully elucidated or understood.

Polyethylene glycol reduces inflammation and aberrant crypt foci in carcinogen-initiated rats.

Polyethylene glycol 8000 inhibits the formation of tumors and of aberrant crypt foci (ACF) in carcinogen-initiated rats. We asked: is the inhibition associated with a reduction of colonic inflammation and an increase in colonic cell permeability? Twenty-eight, male F 344 rats were divided into two groups, 10 control animals and 18 animals initiated with azoxymethane. Nine of the rats in the carcinogen-initiated group were given a diet with 5% PEG 8000 in an AIN-93 based, high fat diet. The other nine, and the control group received the diet without the addition of PEG. Nine weeks later, the rats receiving the diet containing PEG had a 43% reduction in ACF (P<0.001) compared with the carcinogen-initiated rats on the control diet, a result confirming earlier observations that PEG inhibits colon carcinogenesis. The animals receiving the diet containing PEG also had a 10-fold reduction in fecal granulocyte marker protein (GMP) (P<0.001) compared with both the carcinogen-treated and the control animals. PEG reduced inflammation below the levels of carcinogen-treated and of untreated animals. Fecal water from the rats receiving PEG did not reduce transepithelial resistance of, or manitol flux through, human Caco-cells grown as monolayers in vitro. PEG may reduce colon carcinogenesis through a mechanism involving colonic inflammation.

Effects of fermentation products of pro- and prebiotics on trans-epithelial electrical resistance in an in vitro model of the colon.

Evidence from in vivo and in vitro studies suggests that the consumption of pro- and prebiotics may inhibit colon carcinogenesis; however, the mechanisms involved have, thus far, proved elusive. There are some indications from animal studies that the effects are being exerted during the promotion stage of carcinogenesis. One feature of the promotion stage of colorectal cancer is the disruption of tight junctions, leading to a loss of integrity across the intestinal barrier. We have used the Caco-2 human adenocarcinoma cell line as a model for the intestinal epithelia. Trans-epithelial electrical resistance measurements indicate Caco-2 monolayer integrity, and we recorded changes to this integrity following exposure to the fermentation products of selected probiotics and prebiotics, in the form of nondigestible oligosaccharides (NDOs). Our results indicate that NDOs themselves exert varying, but generally minor, effects upon the strength of the tight junctions, whereas the fermentation products of probiotics and NDOs tend to raise tight junction integrity above that of the controls. This effect was bacterial species and oligosaccharide specific. Bifidobacterium Bb 12 was particularly effective, as were the fermentation products of Raftiline and Raftilose. We further investigated the ability of Raftilose fermentations to protect against the negative effects of deoxycholic acid (DCA) upon tight junction integrity. We found protection to be species dependent and dependent upon the presence of the fermentation products in the media at the same time as or after exposure to the DCA. Results suggest that the Raftilose fermentation products may prevent disruption of the intestinal epithelial barrier function during damage by tumor promoters.

Effect of white versus red meat on endogenous N-nitrosation in the human colon and further evidence of a dose response.

N-nitroso compounds are found in the colon and are formed endogenously because amines and amides are produced by bacterial decarboxylation of amino acids in the large gut. They can be N-nitrosated in the presence of a nitrosating agent. To test the hypothesis that increased nitrogenous residues from red meat would increase endogenous N-nitrosation, thus accounting for the epidemiologic association between red meat consumption and colorectal cancer, we fed increased levels of red meat and measured apparent total N-nitroso compounds (ATNCs) in fecal samples in a series of studies of volunteers maintained under controlled conditions. A result of these studies is that we have shown a consistent dose response to red meat consumption. Fiber, in the form of vegetables, bran or resistant starch, does not reduce the level of ATNCs formed, although transit time is reduced and fecal weight are increased. Here we show that the equivalent amount (420-600 g) of meat as white meat has no effect on fecal ATNCs in 12 volunteers (P = 0.338). At dosages of 0, 60, 120, 240 and 420 g of red meat/d, mean levels of ATNC output are highly correlated with dose of meat: for concentration ATNC versus dose of meat in g/d, r = 0.972, beta = 0.252 ng/g (SE 0.035); for total ATNC output versus dose of meat in g/d, r = 0.963, beta = 2.605 microg/d (SE 0.419). The effects of nonmeat protein and of heme on increased N-nitrosation and the genotoxic effects of the ATNCs produced are presently being investigated.

Effect of vegetables, tea, and soy on endogenous N-nitrosation, fecal ammonia, and fecal water genotoxicity during a high red meat diet in humans.

Red meat increases colonic N-nitrosation, and this may explain the positive epidemiological relationship between red meat intake and colorectal cancer risk. Vegetables, tea, and soy have been shown to block N-nitroso compound (NOC) formation and are associated with protection against colorectal cancer. To determine whether these supplements affect fecal NOC excretion during consumption of a high red meat (420 g/day) diet, 11 male volunteers were studied over a randomized series of 15-day dietary periods. Seven of these subjects completed a further dietary period to test the effects of soy (100 g/day). Soy significantly suppressed fecal apparent total NOC (ATNC) concentration (P = 0.02), but supplements of vegetables (400 g/day as 134 g broccoli, 134 g brussels sprouts, and 134 g petits pois) and tea extract (3 g/day) did not affect mean levels of fecal ATNC, nitrogen and ammonia excretion, and fecal water genotoxicity. However, fecal weight was increased (P < 0.001) and associated with reduced transit time (r = 0.594, P < 0.0001), so that contact between ATNC, nitrite, and ammonia and the large bowel mucosa would have been reduced. Longer transit times were associated with elevated fecal ATNC concentrations (r = 0.42, P = 0.002). Fecal nitrite was significantly suppressed during the tea supplement compared with the meat-only (P = 0.0028) and meat + vegetables diets (P = 0.005 for microgram NO2/g).