Differences Between the Intestinal Lumen Microbiota of Aberrant Crypt Foci (ACF)-Bearing and Non-bearing Rats.

BACKGROUND: Multiple factors including host-microbiota interaction could contribute to the conversion of healthy mucosa to sporadic precancerous lesions. An imbalance of the gut microbiota may be a cause or consequence of this process. AIM: The goal was to investigate and analyze the composition of gut microbiota during the genesis of precancerous lesions of colorectal cancer. METHODS: To analyze the composition of gut microbiota in the genesis of precancerous lesions, a rat model of 1, 2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) was established. The feces of these rats and healthy rats were collected for 16S rRNA sequencing. RESULTS: The diversity and density of the rat intestinal microbiota were significantly different between ACF-bearing and non-bearing group. ACF were induced in rats treated with DMH and showed increased expression of the inflammatory cytokines IL-6, IL-8, and TNF-α. Firmicutes was the most predominant phylum in both ACF-bearing and non-bearing group, followed by Bacteroidetes. Interestingly, although the density of Bacteroidetes decreased from the fifth week to the 17th week in both groups, it was significantly reduced in ACF-bearing group at the 13th week (P < 0.01). At the genus level, no significant difference was observed in the most predominant genus, Lactobacillus. Instead, Bacteroides and Prevotella were significantly less abundant (P < 0.01), while Akkermansia was significantly more abundant (P < 0.05) in ACF-bearing group at the 13th week. CONCLUSION: Imbalance of the intestinal microbiota existed between ACF-bearing and non-bearing rats, which could be used as biomarker to predict the genesis of precancerous lesions in the gut.

Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet.

The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat Western diet. To test the hypothesis that a high-fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or an HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1 and inducible nitric oxide synthase in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short-chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that an HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.

Exacerbation of colon carcinogenesis by Blastocystis sp.

Colorectal cancer (CRC) is one the most commonly diagnosed cancers worldwide and the number is increasing every year. Despite advances in screening programs, CRC remains as the second leading cause of cancer deaths in the United States. Oxidative stress plays an important role in the molecular mechanisms of colorectal cancer (CRC) and has been shown to be associated with Blastocystis sp., a common intestinal microorganism. In the present study, we aimed to identify a role for Blastocystis sp. in exacerbating carcinogenesis using in vivo rat model. Methylene blue staining was used to identify colonic aberrant crypt foci (ACF) and adenomas formation in infected rats whilst elevation of oxidative stress biomarker levels in the urine and serum samples were evaluated using biochemical assays. Histological changes of the intestinal mucosa were observed and a significant number of ACF was found in Blastocystis sp. infected AOM-rats compared to the AOM-controls. High levels of urinary oxidative indices including advanced oxidative protein products (AOPP) and hydrogen peroxide were observed in Blastocystis sp. infected AOM-rats compared to the uninfected AOM-rats. Our study provides evidence that Blastocystis sp. has a significant role in enhancing AOM-induced carcinogenesis by resulting damage to the intestinal epithelium and promoting oxidative damage in Blastocystis sp. infected rats.

Structural shift of gut microbiota during chemo-preventive effects of epigallocatechin gallate on colorectal carcinogenesis in mice.

AIM: To investigate the effect of epigallocatechin gallate (EGCG) on structural changes of gut microbiota in colorectal carcinogenesis. METHODS: An azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis mouse model was established. Forty-two female FVB/N mice were randomly divided into the following three groups: group 1 (10 mice, negative control) was treated with vehicle, group 2 (16 mice, positive control) was treated with AOM plus vehicle, and group 3 (16 mice, EG) was treated with AOM plus EGCG. For aberrant crypt foci (ACF) evaluation, the colons were rapidly took out after sacrifice, rinsed with saline, opened longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution before being stained with 0.2% methylene blue in saline. For tumor evaluation, the colon was macroscopically inspected and photographed, then the total number of tumors was enumerated and tumor size measured. For histological examination, the fixed tissues were paraffin-embedded and sectioned at 5 mm thickness. Microbial genomic DNA was extracted from fecal and intestinal content samples using a commercial kit. The V4 hypervariable regions of 16S rRNA were PCR-amplified with the barcoded fusion primers. Using the best hit classification option, the sequences from each sample were aligned to the RDP 16S rRNA training set to classify the taxonomic abundance in QIIME. Statistical analyses were then performed. RESULTS: Treatment of mice with 1% EGCG caused a significant decrease in the mean number of ACF per mouse, when compared with the model mice treated with AOM/DSS (5.38 ± 4.24 vs 13.13 ± 3.02, P < 0.01). Compared with the positive control group, 1% EGCG treatment dependently decreased tumor load per mouse by 85% (33.96 ± 6.10 vs 2.96 ± 2.86, respectively, P < 0.01). All revealed that EGCG could inhibit colon carcinogenesis by decreasing the number of precancerous lesions as well as solid tumors, with reduced tumor load and delayed histological progression of CRC. During the cancerization, the diversity of gut microbiota increased, potential carcinogenic bacteria such as Bacteroides were enriched, and the abundance of butyrate-producing bacteria (Clostridiaceae, Ruminococcus, etc.) decreased continuously. In contrast, the structure of gut microbiota was relatively stable during the intervention of EGCG on colon carcinogenesis. Enrichment of probiotics (Bifidobacterium, Lactobacillu, etc.) might be a potential mechanism for EGCG's effects on tumor suppression. Via bioinformatics analysis, principal coordinate analysis and cluster analysis of the tumor formation process, we found that the diversity of gut microbiota increased in the tumor model group while that in the EGCG interfered group (EG) remained relatively stable. CONCLUSION: Gut microbiota imbalance might be a potential mechanism for the prevention of malignant transformation by EGCG, which is significant for diagnosis, treatment, prognosis evaluation, and prevention of colorectal cancer.

Novel Combination of Prebiotics Galacto-Oligosaccharides and Inulin-Inhibited Aberrant Crypt Foci Formation and Biomarkers of Colon Cancer in Wistar Rats.

The selectivity and beneficial effects of prebiotics are mainly dependent on composition and glycosidic linkage among monosaccharide units. This is the first study to use prebiotic galacto-oligosaccharides (GOS) that contains ß-1,6 and ß-1,3 glycosidic linkages and the novel combination of GOS and inulin in cancer prevention. The objective of the present study is to explore the role of novel GOS and inulin against various biomarkers of colorectal cancer (CRC) and the incidence of aberrant crypt foci (ACF) in a 1,2-dimethyl hydrazine dihydrochloride (DMH)-induced rodent model. Prebiotic treatments of combined GOS and inulin (57 mg each), as well as individual doses (GOS: 76-151 mg; inulin 114 mg), were given to DMH-treated animals for 16 weeks. Our data reveal the significant preventive effect of the GOS and inulin combination against the development of CRC. It was observed that inhibition of ACF formation (55.8%) was significantly (p ≤ 0.05) higher using the GOS and inulin combination than GOS (41.4%) and inulin (51.2%) treatments alone. This combination also rendered better results on short-chain fatty acids (SCFA) and bacterial enzymatic activities. Dose-dependent effects of prebiotic treatments were also observed on cecum and fecal bacterial enzymes and on SCFA. Thus, this study demonstrated that novel combination of GOS and inulin exhibited stronger preventive activity than their individual treatments alone, and can be a promising strategy for CRC chemoprevention.

Effect of Dietary-Resistant Starch on Inhibition of Colonic Preneoplasia and Wnt Signaling in Azoxymethane-Induced Rodent Models.

Dietary fiber has been reported to prevent preneoplastic colon lesions. The aim of this study was to determine the effect of resistant starches, novel dietary fibers, on the development of colonic preneoplasia and Wnt signaling in azoxymethane (AOM)-treated rats and mice fed resistant starches at 55% of the diet after AOM treatment. Another objective was to determine the effect of resistant starches on the development of preneoplasia in rats treated with antibiotics (Ab), administered between AOM treatment and resistant starch feeding. Diets containing resistant starches, high-amylose (HA7), high-amylose-octenyl succinic anhydride (OS-HA7), or high-amylose-stearic acid (SA-HA7) were compared with control cornstarch (CS). The resistant starch content of the diets did not alter the yield of colonic lesions but animals treated with AOM and fed the diet with the highest resistant starch content, SA-HA7 developed the highest average aberrant crypt foci (ACF) per animal. Mice fed the OS-HA7 diet had decreased expression of some upstream Wnt genes in the colonic crypts. This study suggests that further research is needed to determine if resistant starch impacts colon carcinogenesis in rodents.

Hydrolysed inulin alleviates the azoxymethane-induced preneoplastic aberrant crypt foci by altering selected intestinal microbiota in Sprague-Dawley rats.

Context Inulin, a non-digestible carbohydrate isolated from Helianthus tuberosus L. (Asteraceae), has been shown to alter the gut beneficial bacteria including Lactobacillus spp. and Bifidobacteria. Inulin also influences the activities of intestinal microbiota that could prevent the colon cancer development. Objective This study determines the effect of hydrolysed inulin with different degrees of polymerisation on alteration of intestinal microbiota and their activities on azoxymethane (AOM)-induced preneoplastic aberrant crypt foci (ACF) in rats. Materials and methods Seventy-two male Sprague-Dawley rats were randomly divided into six groups (three control and three AOM-treated groups) and the animal were fed with either a normal diet or diet containing 10% of long-chain inulin (InuL) or short-chain inulin (InuS), respectively, for 17 weeks. Colon cancer was induced in rats by injecting AOM subcutaneously at the 8th and 9th week of the study period. At the end of the experiment, cecal contents of rats were examined for selected microbiota, organic acids, putrefactive compounds and microbial enzymes. ACF formation was microscopically examined. Results The inulin diets significantly increased the weight and decreased the pH of the caecal content. The rats fed with InuL-supplemented diet showed approximately 2.9- and 6.8-fold increases in the biomass of Lactobacillus spp. and Bifidobacteria, respectively. Naive and AOM-treated rats fed with inulin-supplemented diet showed ∼1.3- and ∼2.2-fold decreases in the biomass of Escherichia coli and Salmonella enterica serovar Typhi, respectively. Inulins significantly decreased the colonic concentration of phenol, p-cresol and indole. Reduction in the activity of microbial enzymes such as ß-glucuronidase, azoreductase and nitroreductase were observed in inulin-treated animals. Reduction in the ACF formation has been observed in inulin-treated groups. Discussion and conclusion The present study demonstrates that dietary administration of inulin reduces the formation of preneoplastic lesions in the colon, possibly by altering the microecology and microbial activities on carcinogenesis.

Effect of prebiotics on biomarkers of colorectal cancer in humans: a systematic review.

Prebiotics may prevent colorectal cancer (CRC) development in humans by modifying the composition or activity of the colorectal microflora. Epidemiologic and animal studies have shown a reduction in CRC or CRC biomarkers after the administration of prebiotics. Studies using indirect chemical biomarkers of CRC in humans, however, gave mixed results. Recently, human studies measuring direct physical indices of CRC risk after prebiotic consumption have been published. The purpose of this review is to summarize those studies to provide recommendations for the use of prebiotics in CRC risk reduction. A PubMed search was conducted, revealing nine studies. One tested lactulose, two evaluated a blend of oligofructose and inulin, and six measured resistant starch. Lactulose reduced adenoma recurrence, while resistant starch had no effect on adenoma or CRC development. Crypt mitotic location, gene expression, and DNA methylation were somewhat improved after resistant starch consumption. No changes in cell proliferation and apoptosis, crypt morphology, or aberrant crypt foci were found. More human studies measuring physical changes to the gut are needed.

Protective effect of lactofermented beetroot juice against aberrant crypt foci formation and genotoxicity of fecal water in rats.

The aim of the study was to investigate the effects of beetroot juice fermented by Lactobacillus brevis 0944 and Lactobacillus paracasei 0920 (FBJ) on carcinogen induction of aberrant crypt foci (ACF) in rat colon. N-Nitroso-N-methylurea (MNU) was used as carcinogen, which was administrated intragastrically at a dose of 50 mg/kg on the 23rd and 26th day of the experiment. Additionally, we investigated the cytotoxicity and genotoxicity of fecal water from experimental animals in the Caco 2 cell line, evaluated by MTT/NRU tests and the comet assay, respectively, as well as by the count of bacteria adhered to colon epithelium assessed by fluorescence in situ hybridization and DAPI staining. The experimental rats were divided into four groups based on diet type: basal diet, basal diet supplemented with FBJ, basal diet and MNU treatment, and basal diet supplemented with FBJ and MNU treatment. FBJ significantly reduced the number of ACF in MNU-treated rats (from 55±18 to 21±6). Moreover, the number of extensive aberrations (more than 4 crypts in a focus) decreased from 45±21 to 7±4. Fecal water obtained from rats fed with an MNU-containing diet induced pronounced cytotoxic and genotoxic effects in Caco 2 cells, but FBJ supplementation of the diet abolished these effects. The presence of FBJ in the diet significantly increased the count of bacteria, including Lactobacillus/Enterococcus, adhered to colonic epithelium. In conclusion, supplementation of the diet with lactofermented beetroot juice may provide protection against precancerous aberrant crypt formation and reduce the cytotoxic and genotoxic effects of fecal water.