Expression of LGR-5, MSI-1 and DCAMKL-1, putative stem cell markers, in the early phases of 1,2-dimethylhydrazine-induced rat colon carcinogenesis: correlation with nuclear ß-catenin.

BACKGROUND: Colon cancer stem cells may drive carcinogenesis and account for chemotherapeutic failure. Although many markers for these cells have been proposed, there is no complete agreement regarding them, nor has their presence in the early phases of carcinogenesis been characterized in depth. METHODS: The expression of the putative markers LGR-5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), MSI-1 (Musashi-1) and DCAMKL-1 (doublecortin and calcium/calmodulin-dependent protein kinase-like-1) was studied in normal colon mucosa (NM), in the precancerous lesions Mucin Depleted Foci (MDF) and in macroscopic tumours (adenomas) of 1,2-dimethylhydrazine-treated rats. Co-localization between these markers and nuclear ß-catenin (NBC), an attributed feature of cancer stem cells, was also determined. Moreover, since PGE2 could increase NBC, we tested whether short-term treatment with celecoxib, a COX-2 inhibitor (2 weeks, 250 ppm in the diet) could reduce the expression of these markers. RESULTS: LGR-5 expression in NM was low (Labelling Index (LI): 0.22 ± 0.03 (means ± SE)) with positive cells located mainly at the base of the crypts. Compared to NM, LGR-5 was overexpressed in MDF and tumours (LI: 4.7 ± 2.0 and 2.9 ± 1.0 in MDF and tumours, respectively, P<0.01 compared to NM). DCAMKL-1 positive cells, distributed along the length of normal crypts, were reduced in MDF and tumours. Nuclear expression of MSI-1, located mainly at the base of normal crypts, was not observed in MDF or tumours. In both MDF and tumours, few cells co-expressed LGR-5 and NBC (LI: 1.0 ± 0.3 and 0.4 ± 0.2 in MDF and tumours, respectively). Notwithstanding the lower expression of DCAMKL-1 in tumours, the percentage of cells co-expressing DCAMKL-1 and NBC was higher than in NM (LI: 0.5 ± 0.1 and 0.04 ± 0.02 in tumours and NM, respectively). MSI-1 and NBC co-localization was not observed. Celecoxib did not reduce cells co-expressing LGR-5 and NBC. CONCLUSIONS: Based on its prevalent localization at the base of normal crypts, as expected for stem cells, and on the overexpression in precancerous lesions and tumours, we support LGR-5, but not MSI-1 or DCAMKL-1, as putative neoplastic stem cell marker. In both MDF and tumours, we identified LGR-5-positive cells co-expressing NBC which could be a subpopulation with the highest stem cell features.

Sustained proliferation and resistance to apoptosis after a cytotoxic insult are early alterations in rat colon carcinogenesis.

To study the early alterations in carcinogenesis, we determined apoptosis and proliferation in rat mucin depleted foci (MDF), precancerous lesions in the colon under basal conditions and 24 h after treatment with 1,2-dimethylhydrazine (DMH), which induces apoptosis in the colon. Spontaneous apoptosis in MDF was higher than in normal mucosa (Apoptotic Index was 1.61 ± 0.30 and 0.21 ± 0.02 in MDF and normal mucosa, respectively, mean ± SE, p < 0.05). DMH (30 and 75 mg/kg) increased apoptosis in both normal mucosa and MDF (up to 20 times higher compared to basal levels in normal mucosa, but only two times in MDF). MDF had a higher and deregulated pattern of proliferation along the crypt compared to normal mucosa. After DMH, proliferation in normal mucosa was significantly depressed, but it did not vary in MDF. Survivin-Birc5 regulating apoptosis and proliferation was significantly over-expressed (RT-qPCR and immunohistochemistry experiments) in MDF vs. normal mucosa, but did not vary in response to DMH. The expression of the pro-apoptotic protein Bak did not vary in normal mucosa and MDF. Since inflammation is present in MDF, which may hamper apoptosis, we studied the effect of pre-treatment with aspirin (600 ppm in the diet for 10 days). No significant effects of aspirin were observed. In conclusion, MDF had a higher spontaneous apoptosis and proliferation coupled with a reduced response to apoptotic stimuli from cytotoxic compounds. Survivin over-expression in MDF indicates that this is an early event in colon carcinogenesis and suggests that down-regulation of Survivin may represent a strategy for cancer prevention.

Arabinoxylan-oligosaccharides (AXOS) reduce preneoplastic lesions in the colon of rats treated with 1,2-dimethylhydrazine (DMH).

BACKGROUND: Prebiotics are non-digestible compounds that beneficially affect the host by stimulating the growth and/or activity of one or a limited number of resident colonic bacteria in the gut. Reported beneficial effects of prebiotics include reduced gut infections, better absorption of minerals, and notably, antitumorigenic effects. Arabinoxylan (AX)-oligosaccharides (AXOS) have been suggested to exert prebiotic effects in the gut, but their effect on colon carcinogenesis has not been studied so far. AIM OF THE STUDY: To test the effect of AXOS in a rat colon carcinogenesis model. METHODS: We determined the occurrence of two types of preneoplastic lesions [aberrant crypt foci (ACF) and mucin depleted foci (MDF)] in the colon of rats treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) and fed either a control diet or a diet containing AXOS (4.8% w/w) (15 rats in each group). RESULTS: Thirteen weeks after DMH treatment, MDF counts were significantly lower in the entire colon of AXOS fed rats (MDF/colon were 7.5 +/- 0.6 and 5.5 +/- 0.6, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). Although the number of ACF in the entire colon was not significantly different between Control and AXOS fed rats, AXOS fed rats had significantly fewer ACF in the distal part of the colon than Control group rats (ACF/distal colon were 135.5 +/- 15 and 84.4 +/- 11, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). CONCLUSIONS: The present study shows that dietary intake of AXOS by rats reduces the occurrence of two types of preneoplastic lesions, thus suggesting a chemopreventive effect on colon carcinogenesis that should be confirmed in a long-term carcinogenesis experiment.

Mucin-depleted foci show strong activation of inflammatory markers in 1,2-dimethylhydrazine-induced carcinogenesis and are promoted by the inflammatory agent sodium dextran sulfate.

Mucin-depleted foci (MDF), formed by dysplastic crypts devoid of mucins, have been identified in the colon of carcinogen-treated rodents and in humans at high risk for colon cancer. The lack of the protective layer of mucus may cause inflammation which has been linked to colon carcinogenesis, therefore, the expression of markers such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (i-NOS) and macrophage infiltration was studied with immunohistochemistry (IH) in MDF harvested from F344 rats treated with the colon carcinogen 1,2-dimethylhydrazine (DMH). The same determinations were performed in aberrant crypt foci (ACF) and, at a later time point, in tumours. A dramatic increase in COX-2, i-NOS and macrophage infiltration was observed in MDF but ACF showed a moderate increase compared with the paired normal mucosa. Tumours were positive for all the markers. RT-PCR experiments demonstrated that i-NOS RNA expression was increased in a set of MDF confirming the results obtained with immunohistochemistry. In an inflammation-cancer experimental model [mice treated with azoxymethane (AOM) and dextran sodium sulphate (DSS)], we observed that DSS-induced inflammation promoted MDF in a dose-dependent manner, whereas ACF were not affected. In conclusion, we report here for the first time a strong activation of the inflammatory process in MDF, which may contribute to the further progression of MDF to tumours.

Reduction of colonic inflammation in HLA-B27 transgenic rats by feeding Marie Ménard apples, rich in polyphenols.

Inflammatory bowel diseases (IBD) are immunomediated ailments affecting millions of individuals. Although diet is regarded as an important factor influencing IBD, there are no accepted dietary recommendations presently available. We administered 7.6 % lyophilised apples obtained from two cultivars (Golden Delicious and Marie Ménard, low and high in polyphenols, respectively) to HLA-B27 transgenic rats which develop spontaneous IBD. After 3 months feeding, rats fed Marie Ménard apples had reduced myeloperoxidase activity (3.6 (sem 0.3) v. 2.2 (sem 0.2) U/g tissue; P < 0.05) and reduced cyclo-oxygenase-2 (P < 0.05) and inducible NO synthase gene expression (P < 0.01) in the colon mucosa and significantly less diarrhoea (P < 0.05), compared with control rats. Cell proliferation in the colon mucosa was reduced significantly by feeding Golden Delicious apples, with a borderline effect of Marie Ménard apples. Gene expression profiling of the colon mucosa, analysed using the Whole Rat Genome 4 x 44 K Agilent Arrays, revealed a down-regulation of the pathways of PG synthesis, mitogen-activated protein kinase (MAPK) signalling and TNFalpha-NF-kappaB in Marie Ménard-fed rats. In the stools of the animals of this group we also measured a significant reduction of bacteria of the Bacteriodes fragilis group. In conclusion, the administration of Marie Ménard apples, rich in polyphenols and used at present only in the manufacturing of cider, ameliorates colon inflammation in transgenic rats developing spontaneous intestinal inflammation, suggesting the possible use of these and other apple varieties to control inflammation in IBD patients.

Frequent mutation of Apc gene in rat colon tumors and mucin-depleted foci, preneoplastic lesions in experimental colon carcinogenesis.

Mucin-depleted foci (MDF) are microscopic dysplastic lesions induced in the colon of rodents by specific colon carcinogens. Most MDF show Wnt pathway activation, whereas only a subset shows mutations in the Ctnnb1 gene, coding for beta-catenin. Because Apc is a member of the Wnt pathway and the most frequent mutated gene in human colon cancer, we tested whether MDF harbor Apc mutations. F344 rats were treated twice with 150 mg/kg of 1,2-dimethylhydrazine. After 15 or 28 weeks, MDF, aberrant crypt foci (ACF), and tumors were collected. We screened a segment of the Apc gene comprising the region homologous to the mutation cluster region (MCR) of human APC, which frequently shows mutations in experimental colon tumors. Mutations were identified by PCR amplification and sequencing in 6:24 MDF (25%), 7:23 tumors (30%), 0:24 ACF (0%). Most of the mutations (92%) in MDF and tumors were localized in a region upstream from the MCR. All mutations were single-base substitutions and mainly formed by G:C-->A:T and C:G-->T:A transitions. The pattern of nucleotide changes was similar in MDF and tumors, and, interestingly, the same mutation in codon 1047 was found in two MDF and in three tumors. Four out of the six mutations found in MDF were nonsense mutations, and two were missense. All mutations in tumors determined a protein truncation. These results show that Apc mutations are present in MDF with a frequency similar to that of tumors, strengthening the evidence that they are precancerous lesions in colon carcinogenesis.

K-ras mutations and mucin profile in preneoplastic lesions and colon tumors induced in rats by 1,2-dimethylhydrazine.

K-ras and mucin profile variations, associated with intestinal carcinogenesis, were studied in the preneoplastic lesions, mucin-depleted foci (MDF) and aberrant crypt foci (ACF), and in colonic tumors induced in rats by 1,2-dimethylhydrazine (DMH). The frequency of lesions with K-ras mutations was 23% (3/13), 5.5% (1/18) and 100% (14/14) in MDF, tumors and ACF, respectively. Two of three MDF mutated in K-ras also carried a missense mutation in Apc. We also tested the expression of MUC2, a mucin abundantly expressed in normal colon and M1/MUCA5C, up-regulated in colon carcinogenesis, using immunohistochemistry. MDF and tumors showed a dramatic reduction in the expression of MUC2, whereas ACF showed only a slight reduction. The expression of M1/MUC5AC was almost absent in normal mucosa, but was increased in all the lesions (MDF, tumors and ACF). The expression of the intestinal trefoil factor (ITF), a marker of goblet cell lineage, was reduced in MDF and tumors compared to normal mucosa but not in ACF. In conclusion, although K-ras mutations are present in all ACF, they are less frequent in MDF and tumors; M1/MUC5AC is a marker associated with all preneoplastic events while the reduction of MUC2 and ITF expression is selectively associated with more advanced lesions such as MDF and tumors.

No effects of olive oils with different phenolic content compared to corn oil on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

BACKGROUND: Some epidemiological and experimental studies suggest that olive oil, despite its elevated caloric content, may have protective activity against colon cancer, partially due to its phenolic content. However, little experimental evidence exists to support this claim in vivo. AIM OF THE STUDY: To test the effect of olive oils with different phenolic content in a well-characterized model of colon carcinogenesis, comparing them with corn oil (CO). METHODS: F344 rats were fed AIN-76 based diets for the entire experimental period; the diets contained 23% (w/w) of lipids from three different sources: extra-virgin olive oil rich in phenolic compounds (EV), rectified olive oil (ROO) with the same fatty acid composition but devoid of phenolic compounds and CO as a control diet. One week later, rats were induced with 1,2-dimethylhydrazine (DMH) (150 mg/kg b.w. x 2 times) to measure preneoplastic lesions (aberrant crypt foci (ACF) and mucin depleted foci (MDF)) and tumours in the intestine. RESULTS: Thirteen weeks after DMH, the numbers of ACF and MDF were similar in the different groups (ACF/colon were 344.9 +/- 27.0, 288.6 +/- 28.5 and 289.8 +/- 21.4 in CO, EV and ROO groups, respectively, means +/- SE; MDF/colon were 8.83 +/- 1.2, 8.41 +/- 1.5 and 8.75 +/- 1.6 in CO, EV and ROO groups, respectively, means +/- SE). Thirty-two weeks after DMH, the incidence of tumours (rats with tumours/rats in the group) did not differ among the different groups (20/21, 18/19 and 20/20 in the CO, EV, and ROO groups, respectively). Similarly, the number of tumours/ rat in the colorectum (both adenomas and cancers) was not different in the three different groups (2.33 +/- 0.26, 2.42 +/- 0.41 and 2.25 +/- 0.40 in CO, EV and ROO groups, respectively, means +/- SE). CONCLUSIONS: Olive oil, irrespective of its phenolic content, does not affect DMH-induced colon carcinogenesis in F344 rats compared with CO.

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.

Identification of mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with carcinogenesis.

We tested the association between aberrant crypt foci (ACF) and tumor induction by feeding azoxymethane-induced rats (15 mg/kg x 2, s.c.) with synbiotics (Raftilose Synergy 1, a derivative of inulin, 10% of the diet, along with lactobacilli and bifidobacteria). After 16 weeks of feeding, synbiotics significantly increased ACF multiplicity. On the contrary, after 32 weeks, synbiotics significantly decreased intestinal tumors. When the same unsectioned colon used for ACF determination was stained with high-iron diamine Alcian blue, foci of crypts with scarce or absent mucins were identified. We defined these lesions as mucin-depleted foci (MDF), and they were visible in all azoxymethane-treated rats and correlated with tumor induction (MDF/colon: 8.2 +/- 0.9 and 3.8 +/- 0.9 in controls and synbiotic-fed rats, respectively, P < 0.01; crypts/MDF: 12.2 +/- 2 and 6.4 +/- 1 in controls and synbiotic-fed rats, respectively, P < 0.05, means +/- SE, n = 7). There were fewer MDF/colon than ACF, and they were histologically more dysplastic than mucinous lesions identified as ACF in high-iron diamine Alcian blue-stained colon. In conclusion, MDF may be premalignant lesions that predict colon carcinogenesis.

Mucin depleted foci, colonic preneoplastic lesions lacking Muc2, show up-regulation of Tlr2 but not bacterial infiltration.

Mucin depleted foci (MDF) are precancerous lesions of the colon in carcinogen-treated rodents and humans at high risk. Since MDF show signs of inflammation we hypothesized that the defective mucous production would expose them to the risk of being penetrated by intestinal bacteria, which can be sensed by Toll-like receptors (Tlrs) and activate inflammatory pathways. To verify this hypothesis we tested the expression of 84 genes coding for Tlrs and associated pathways using RT-qPCR in MDF (n = 7) from 1,2-dimethylhydrazine (DMH)-treated rats. Among the 84 tested genes, 26 were differentially expressed in MDF with 5 genes significantly up-regulated and 21 down-regulated when compared to the normal mucosa. Tlr2, as well as other downstream genes (Map4k4, Hspd1, Irak1, Ube2n), was significantly up-regulated. Among the genes regulating the NFkB pathway, only Map4k4 was significantly up-regulated, while 19 genes were not varied and 6 were down-regulated. Tlr2 protein was weakly expressed both in normal mucosa and MDF. To determine whether inflammation observed in MDF could be caused by bacteria contacting or infiltrating crypts, we performed fluorescence in situ hybridization (FISH) experiments with a rRNA universal bacterial probe. None of the 21 MDF tested, showed bacteria inside the crypts, while among the colonic tumors (n = 15), only one had very few bacteria on the surface and on the surrounding normal mucosa. In conclusion, the up-regulation of Tlr2 in MDF, suggests a link between this receptor and carcinogenesis, possibly related to a defective barrier function of these lesions. The data of FISH experiments do not support the hypothesis that inflammation in MDF and tumors is stimulated by bacterial infiltration.

Marie Ménard apples with high polyphenol content and a low-fat diet reduce 1,2-dimethylhydrazine-induced colon carcinogenesis in rats: effects on inflammation and apoptosis.

Inflammation may increase cancer risk, therefore, we studied whether polyphenol-rich Marie Ménard (MM) apples with reported anti-inflammatory activity prevent 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats and, likewise whether high-fat (HF) diet promoting carcinogenesis, may affect inflammation. DMH-induced rats were fed for 15 weeks with: an HF diet (23% corn oil w/w); an HF diet containing 7.6% w/w lyophilized MM (apple diet (AD)); a low-fat (LF) diet and an HF diet containing piroxicam (PXC) (0.01% w/w) as control. Mucin depleted foci (MDF), precancerous lesions in the colon, were dramatically reduced in the AD, LF, and PXC groups compared with the HF. Peritoneal macrophage activation, an index of systemic inflammation, was significantly decreased in the AD, LF, and PXC groups. TNF-α, iNOS, IL-1ß, IL-6 m-RNA expression in the colon, as well as CD68 cells and plasmatic PGE2 were lower in the AD, but not in the LF group. Apoptosis in the MDF of both the AD and LF-fed rats was significantly higher than in HF rats. In conclusion, AD has a strong chemopreventive effect, reducing inflammation, and increasing apoptosis, while the chemopreventive effect of the LF diet seems mediated mainly by increased apoptosis in MDF.

Identification of mucin depleted foci in the human colon.

Aberrant crypt foci (ACF) originally described in rodents treated with colon-specific carcinogens have been identified also in humans at high risk of colon cancer (CRC) and are extensively used as cancer biomarkers. However, studies documenting the heterogeneity of ACF have questioned their precancerous nature. Recently, we described dysplastic foci depleted of mucins (MDF) in the colon of rats treated with colon-specific carcinogens. Like colon tumors, MDFs show activation of Wnt signaling driven by mutations in the beta-catenin gene and Apc, a key gene in colorectal carcinogenesis. Because MDFs have been identified thus far only in rodents, we wanted to search for similar lesions in humans. Familial adenomatous polyposis (FAP) subjects, carrying germ-line mutations in the APC gene, are at high risk of CRC. Therefore, we first searched for MDF-like lesions in unsectioned colon samples from FAP patients and then in patients with sporadic CRC. MDFs were present in the colon of FAP patients (average of 0.0577 lesions/cm(2)) and at a much lower density in CRC patients (average of 0.0006 lesions/cm(2)). ACFs were also observed in all patients. Histologic preparations of all the MDFs identified in FAP and CRC consisted of microadenomas at variable grades of dysplasia. The occurrence of MDF-like lesions in high-risk patients provides evidence that these lesions have a counterpart in human pathology and, as observed in rodents, may represent the very early stages of CRC.

Mucin-depleted foci are modulated by dietary treatments and show deregulation of proliferative activity in carcinogen-treated rodents.

The correlation between mucin-depleted foci (MDF) and colon carcinogenesis was studied in F344 rats initiated with 1,2-dimethylhydrazine and treated with a chemopreventive regimen (polyethylene glycol, PEG) or with a promoting diet (high-corn oil). High corn oil diet increased MDF, while PEG reduced them. The expression of p27 and p16, inhibitors of cyclin-dependent kinases, which inhibit the progression of the cell cycle, was studied by immunohistochemistry in MDF and in aberrant crypt foci (ACF) of control rats. In both MDF and ACF, the nuclear expression of p27 was markedly reduced, while p16 was reduced to a lower extent. Mitotic activity was higher in MDF and ACF than in normal mucosa of control rats. MDF were also identified in azoxymethane-initiated SWR/J mice. These results further confirm that MDF are preneoplastic lesions and could be useful biomarkers of colon carcinogenesis.

Mucin-depleted foci have beta-catenin gene mutations, altered expression of its protein, and are dose- and time-dependent in the colon of 1,2-dimethylhydrazine-treated rats.

Mucin-depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen-treated rats stained with high-iron diamine alcian blue (HID-AB). In F344 rats treated twice with 150 mg/kg of 1,2-dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg x 2 times) caused a dose-related increase in MDF. Mutations in Ctnnb1 gene codifying for beta-catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID-AB-stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. Beta-catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID-AB. By contrast, nuclear localization of beta-catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. Beta-catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose-dependently by DMH, increase in size with time, have mutations in the beta-catenin gene and marked alterations in beta-catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short-term carcinogenesis experiments.

Effect of polyphenolic extracts from red wine and 4-OH-coumaric acid on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

BACKGROUND: Total polyphenolic extracts from red wine protect against azoxymethane (AOM)-induced colon carcinogenesis in rats. Since red wine contains more than 200 different polyphenolic compounds, it is still unclear which substances are responsible for this effect. AIM OF THE STUDY: We investigated the effect of high molecular weight polyphenols (HMWP), low molecular weight polyphenols (LMWP) and total polyphenolic extracts from red wine (WE) on colon carcinogenesis. We also tested the effect of 4-OH-coumaric acid, a potent phenolic antioxidant present in wine and fruit. METHODS: F344 rats were treated weekly with 1,2-dimethylhydrazine (DMH) (30 mg/kg b.w. subcutaneously x 10 times). One week after the final DMH injection rats were divided into five groups and fed: a) a high fat (HF) diet containing 23% corn oil (w/w), as control or the same basal diet supplemented with b) 0.11 % (w/w) WE; c) 0.027% (w/w) HMWP d) 0.083% (w/w) LMWP or e) 0.1 % (w/w) 4-OH-coumaric acid. The dietary treatments continued until sacrifice, 16 weeks after the last DMH injection. RESULTS: WE treated rats had significantly fewer (p < 0.05) colorectal adenomas than controls, while rats in other treatment groups did not differ significantly from controls (colorectal adenomas/rat were: 2.2 +/- 0.3; 1.4 +/- 0.2; 2.9 +/- 0.5; 2.6 +/- 0.4; 2.3 +/- 0.3; in controls, WE, HMWP, LMWP and 4-OH-coumaric acid groups, respectively; means +/- SE). The mean number of colorectal carcinomas per rat was similar among all experimental groups. Proliferative activity in the normal colon mucosa did not vary among experimental groups. CONCLUSIONS: Total polyphenolic extracts (WE) from red wine, but neither the HMWP nor the LMWP, have some inhibitory effect on the process of colon carcinogenesis by DMH reducing the number of adenomas.

Effect of diets fortified with tomatoes or onions with variable quercetin-glycoside content on azoxymethane-induced aberrant crypt foci in the colon of rats.

BACKGROUND: Onion and tomato are vegetables widely consumed by humans and epidemiological studies show an inverse association between vegetable consumption and colon cancer risk; however, the effect on colon cancer of diets containing high levels of vegetables like onion and tomato are not clear. AIMS OF THE STUDY: To investigate whether tomatoes and onions,with low or high quercetin-glycoside content, could reduce azoxymethane (AOM)-induced Aberrant Crypt Foci (ACF), preneoplastic lesions in the colon of rats. METHODS: Male Fisher 344 rats were fed the following diets: a) high fat (HF) diet (control diet); b) HF diet containing 20 % (w/w) tomatoes with a low quercetin-glycoside content (final concentration in the diet: 5 mg/kg of quercetin aglycone equivalents); c) HF diet containing 20% (w/w) high quercetin-glycoside tomatoes (100 mg/kg final concentration of quercetin aglycone equivalents); d) HF diet containing 20 % (w/w) low quercetin-glycoside onions (14 mg/kg of quercetin aglycone equivalents in the diet); e) HF diet containing 20 % (w/w) high quercetin-glycoside onions (360 mg/kg quercetin aglycone equivalents in the diet). After 2 wks of feeding, all rats were treated twice, 1 wk apart, with AOM (12 mg/kg, s. c.). The dietary treatments continued until sacrifice, 7 wks after the first injection with AOM. RESULTS: ACF induction did not vary in animals fed low or high quercetin-glycoside tomatoes relative to controls. On the contrary, rats fed 20% (w/w) onion-based diets, with low or high quercetin-glycoside content, showed an increase in number, multiplicity and "large" ACF compared to the control group (number of ACF/colon 145 +/- 15 (SE), 255 +/- 11 and 218 +/- 16 in controls, low and high-quercetin-glycoside groups, respectively; p < 0.01). Proliferative activity of the colon did not vary between animals fed control and high quercetin-glycoside tomato diet. The height of the crypts in normal mucosa of rats fed high quercetinglycoside onions was significantly increased compared to control rats (cells/emicrypt 38.4 +/- 1.2 (SE) and 41.3 +/- 0.6 in controls and high quercetin-glycoside onions group, p < 0.05). CONCLUSIONS: None of the diets supplemented with onion or tomato with variable quercetin-glycoside content demonstrated a potential chemopreventive effect on ACF-induction by AOM in rats.

Fecal levels of short-chain fatty acids and bile acids as determinants of colonic mucosal cell proliferation in humans.

We studied the correlation between fecal levels of short-chain fatty acids (SCFA), bile acids (BA), and colonic mucosal proliferation in humans on a free diet. Subjects [n = 43: 27 men and 16 women; 61 +/- 7 and 59 +/- 6 (SE) yr old, respectively] were outpatients who previously underwent resection of at least two sporadic colon polyps. Mucosal proliferation was determined by [3H]thymidine incorporation in vitro in three colorectal biopsies obtained without cathartics and was expressed as labeling index (LI). BA were analyzed in feces by mass spectrometry and SCFA by gas chromatography. We found that increasing levels of BA in feces did not correlate with higher LI. On the contrary, higher levels of SCFA were significantly associated with lower LI in the colonic mucosa (P for trend = 0.02). In conclusion, in humans on a free diet, intestinal proliferation seems to be regulated by the levels of SCFA in feces and not by BA. Because a lower intestinal proliferation is associated with a decreased colon cancer risk, treatments or diets that increase colonic levels of SCFA might be beneficial for colonic mucosa.

Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in rats.

Prebiotics such as fructans, and probiotics such as Lactobacilli or Bifidobacteria, or a combination of prebiotics and probiotics (synbiotics) are thought to be protective against colon cancer. Therefore, we studied whether the prebiotic inulin enriched with oligofructose (Raftilose-Synergy1, briefly, Synergy1, 10% of the diet), probiotics [Bifidobacterium lactis (Bb12) and Lactobacillus rhamnosus (LGG), each at 5x10(8) c.f.u./g diet] or synbiotics (a combination of the two) protect rats against azoxymethane (AOM)-induced colon cancer. Male F344 rats were divided into: Controls; PRE, which were fed a diet containing Synergy1; PRO, fed a diet containing LGG and Bb12; PREPRO, fed a diet containing Synergy1, LGG and BB12. Ten days after beginning the diets, rats were treated with AOM (15 mg/kg s.c. two times); dietary treatments were continued for the entire experiment. Thirty-one weeks after AOM, rats treated with Synergy1 (PRE and PREPRO groups) had a significantly lower (P < 0.001) number of tumours (adenomas and cancers) than rats without Synergy1 (colorectal tumours/rat were 1.9 +/- 1.7, 1.1 +/- 1.1, 2.2 +/- 1.4 and 0.9 +/- 1.2 in Controls, PRE, PRO and PREPRO groups, respectively, means +/- SD). A slight, not significant effect of probiotics in reducing malignant tumours was also observed (P = 0.079). Caecal short-chain fatty acids (SCFA) were higher (P < 0.001) in the groups treated with Synergy1. Apoptosis was increased in the normal mucosa of the PRO group, while no variation was observed in the tumours. Colonic proliferation was lower in the PRE group as compared with Controls. Glutathione S-transferase placental enzyme pi type expression, and to a lesser extent, inducible NO synthase were depressed in the tumours from rats in the PRE and PREPRO groups. Cycloxygenase-2 expression was increased in the tumours of control rats but not in those from PRE, PRO or PREPRO rats. In conclusion, prebiotic administration in the diet decreases AOM-induced carcinogenesis in rats.