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Cruciferous vegetables and glutathione: their effects on colon mucosal glutathione level and colon tumor development in rats induced by DMH.

The effect of a diet containing 10-40% lyophilized cabbage or broccoli as cruciferous vegetable or 10-40% lyophilized potato as noncruciferous vegetable fed for 14 days on the colon mucosal glutathione (GSH) level was studied in male rats. The GSH levels of the duodenum mucosa and the liver were also measured. Cabbage and broccoli enhanced the colon and duodenum mucosal GSH levels in a dose-related manner; potato had no effect. All three vegetables had no effect on the liver GSH level. The effect of GSH on colon tumorigenesis induced by 1,2-dimethylhydrazine (DMH) was also examined in rats. Male Sprague-Dawley rats were injected with DMH (20 mg/kg body wt) weekly for 20 weeks. DMH lowered the colon mucosal GSH level. GSH (100 mg/day/rat) dissolved in the drinking water and given to rats during and after DMH injections had little or no effect on tumor incidence and total number of colon tumors. Tumors were larger in rats that received GSH than in those that received water. This study shows that the colon mucosal GSH level can be enhanced by feeding rats a diet high in cabbage or broccoli and that GSH added to the drinking water did not affect DMH-induced colon tumorigenesis under the experimental conditions used.

1,2-Dimethylhydrazine↗

Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium.

High intake of red meat is associated with increased colon cancer risk. We have shown earlier that this may be due to the high haem content of red meat, because dietary haem increased cytolytic activity of faecal water and colonic epithelial proliferation. Dietary calcium inhibits diet-induced epithelial hyperproliferation. Furthermore, it has been shown that supplemental calcium inhibited the recurrence of colorectal adenomas. Therefore, we studied whether dietary calcium phosphate can exert its protective effects by inhibiting the deleterious effects of haem. In vitro, calcium phosphate precipitated haem and inhibited the haem-induced cytotoxicity. Subsequently, rats were fed diets, differing in haem (0 or 1.3 micromol/g) and calcium phosphate content only (20 or 180 micromol/g). Faeces were collected for biochemical analyses. Cytolytic activity of faecal water was determined from the degree of lysis of erythrocytes by faecal water. Colonic epithelial proliferation was measured in vivo using [(3)H]thymidine incorporation. In rats fed low calcium diets, dietary haem increased cytolytic activity of faecal water (98 +/- 1 versus 1 +/- 1%, P < 0.001) and the concentration of cations in faeces (964 +/- 31 versus 254 +/- 20 micromol/g), when compared with controls. This indicates that dietary haem increased colonic mucosal exposure to luminal irritants. Colonic epithelial proliferation was increased compared with controls (70 +/- 4 versus 48 +/- 8 d.p.m./microg DNA, P < 0.001). This was accompanied by metabolism of the ingested haem and solubilization of haem compounds in the faecal water. A high calcium diet largely prevented this metabolism and solubilization. It also inhibited the haem-induced cytolytic activity of faecal water and increase in faecal cation concentration. In accordance, the haem-induced colonic epithelial hyperproliferation was prevented. We therefore suggest that dietary calcium phosphate acts as a chemopreventive agent in colon carcinogenesis by inhibiting the cytolytic and hyperproliferative effects of dietary haem.

Animals↗

Green vegetables, red meat and colon cancer: chlorophyll prevents the cytotoxic and hyperproliferative effects of haem in rat colon.

Diets high in red meat and low in green vegetables are associated with increased colon cancer risk. This association might be partly due to the haem content of red meat. In rats, dietary haem is metabolized in the gut to a cytotoxic factor that increases colonic cytotoxicity and epithelial proliferation. Green vegetables contain chlorophyll, a magnesium porphyrin structurally analogous to haem. We studied whether green vegetables inhibit the unfavourable colonic effects of haem. First, rats were fed a purified control diet or purified diets supplemented with 0.5 mmol haem/kg, spinach (chlorophyll concentration 1.2 mmol/kg) or haem plus spinach (n = 8/group) for 14 days. In a second experiment we also studied a group that received haem plus purified chlorophyll (1.2 mmol/kg). Cytotoxicity of faecal water was determined with a bioassay and colonic epithelial cell proliferation was quantified in vivo by [methyl-(3)H]thymidine incorporation into newly synthesized DNA. Exfoliation of colonocytes was measured as the amount of rat DNA in faeces. In both studies haem increased cytotoxicity of the colonic contents approximately 8-fold and proliferation of the colonocytes almost 2-fold. Spinach or an equimolar amount of chlorophyll supplement in the haem diet inhibited these haem effects completely. Haem clearly inhibited exfoliation of colonocytes, an effect counteracted by spinach and chlorophyll. Finally, size exclusion chromatography showed that chlorophyll prevented formation of the cytotoxic haem metabolite. We conclude that green vegetables may decrease colon cancer risk because chlorophyll prevents the detrimental, cytotoxic and hyperproliferative colonic effects of dietary haem.

Animals↗

Dietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis.

BACKGROUND: We previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined. RESULTS: Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or WPH vs. CAS) were found. We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH. CONCLUSIONS: The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.

Administration, Oral↗

Are dietary fiber-induced alterations in colonic epithelial cell proliferation predictive of fiber's effect on colon cancer?

Alterations in cell proliferation of the colon have been observed as a result of changes in amount and type of dietary fiber and in relation to risk of developing colon cancer. Although some human observational and intervention studies contribute to the database, most information results from experiments on rodents. Because of numerous contradictory reports linking dietary fiber, cell proliferation, and colon cancer, we undertook a critical review of existing methods in an attempt to explain the inconsistencies. Although there may be some individual types of dietary fiber that protect against chemically induced colon cancer, dietary fiber as a single entity does not appear to afford any consistent protection. Because of significant differences in experimental protocols among laboratories, it is not yet possible to state with certainty that increases in cell proliferation, induced by fiber consumption, are predictive of increased tumorigenesis. Much of what has been observed and interpreted as elevation of risk may simply be normal homeostatic changes in cell proliferation. Even though fermentation to short-chain fatty acids is a mechanistically attractive hypothesis to explain why fiber modulates cytokinetics, data do not consistently support short-chain fatty acids as biological intermediates in risk of colon cancer. The state of the art in this field has not yet progressed to the point where a clear effect of dietary fiber on cytokinetics and colon carcinogenesis can be assessed with any degree of certainty. Additional markers of apoptosis, differentiation, and cell-cell communication may be required for a more accurate analysis of the relation among fiber, cytokinetics, and colon cancer.

Animals↗

Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.

The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.

Animals↗

Association between permeability of the colonic wall and azoxymethane induced cancer of the colon in rats.

OBJECTIVE: To investigate the association between colonic permeability and the development of azoxymethane induced colonic cancer in rats. MATERIAL: Seventy-three male Fischer-Cooper hybrid rats. INTERVENTIONS: Measurement of the concentrations of sodium fluorescein in plasma as an indication of its passage across the bowel wall in control rats, and six weeks and six months after injection of azoxymethane. RESULTS: Forty-seven rats were given azoxymethane, and 26 acted as controls. Sodium fluorescein was instilled into segments of right (n = 46) and left (n = 27) colon and measured in peripheral blood; significantly higher concentrations were recorded after instillation into the left than into the right colon. No tumours developed in the 15 rats that were given azoxymethane and were examined after six weeks. At six months, however, 29 of the remaining 32 had developed 94 macroscopic tumours (range 1-12 tumours/rat), and 89 of these (95%) were in the left colon. CONCLUSION: The greater permeability of the left colon in rats compared with the right may be associated with the higher incidence of carcinomas in the left compared with the right colon.

Animals↗

Lifting of the colon for laparoscopic-assisted colectomy for colon and rectal cancer.

BACKGROUND AND OBJECTIVES: Laparoscopic-assisted colectomy for colon and rectal cancer causes less surgical trauma than does open colectomy. However, current methods are more costly and require highly skilled staff. In addition, the technique for lymphadenectomy has yet to be standardized. We developed a technique that uses a nylon suture to elevate the colon. This method reduces costs without compromising the completeness of the resection. METHODS: Three trocars are introduced and a 1-0 nylon suture is passed into the abdominal cavity and through the mesocolon. The colon is retracted anteriorly and is fixed by this suture to the abdominal wall. The main mesenteric vessels are under tension, and lymph node dissection is performed easily. This method requires only 2 surgeons, an operator, and a scopist, because the colon is fixed to the abdominal wall. In addition, the working space is more stable because the colon is fixed to the abdominal wall. The procedure is relatively independent of the skill of the first assistant. RESULTS: From April 2000 to August 2002, this method was performed in 52 patients. The mean number of dissected lymph nodes was 16.9+/-9.0 (range, 6 to 41). Nine patients had lymph node metastases (17.3%). One patient developed hepatic recurrence; all patients are alive. No complication occurred that was related to lifting the colon. CONCLUSIONS: Using a suture to lift the colon is a useful method for performing laparoscopic-assisted colectomy with lymphadenectomy. This method reduces the number of surgical staff and the expense of the procedure.

Colectomy↗

Effects of dietary fish oil and corn oil on protein kinase C distribution in the rat colon with and without 1,2 dimethylhydrazine treatment and in rat colonic adenocarcinoma.

The activity and subcellular distribution of Protein Kinase C (PKC) was determined in the colons of Sprague-Dawley rats that were fed either a low fat rat chow or rat chow supplemented with 17% corn oil (40% ingested calories as fat). Rats given the high fat diets were either given no carcinogen or treated prior to or subsequent to the initiation of the test diets with 1,2 dimethylhydrazine (DMH). Rats were sacrificed and PKC activity determined in the soluble and particulate fractions of the colonic tissue 13 weeks after the initiation of the diets or DMH treatment, which was before tumor induction. In addition several rats were maintained on their diets until colon tumor formation occurred and PKC activity determined in the colonic tumor and compared to age matched control colonic tissue. In the absence of DMH, fish oil and corn oil equally augmented PKC activity and decreased the ratio of soluble/particulate PKC. With DMH treatment, corn oil augmented PKC as above, but fish oil supplementation resulted in a pattern of PKC activity and distribution more typical of a low fat diet, particularly when fish oil supplementation preceded DMH treatment. PKC activity in DMH induced colonic carcinomas was markedly depressed regardless of the fat source in the diet, when compared to colonic tissue from a non-DMH treated age matched low fat control.

1,2-Dimethylhydrazine↗

Relationship between colonic luminal pH, cell proliferation, and colon carcinogenesis in 1,2-dimethylhydrazine treated rats fed high fiber diets.

The comparative effects of different fibers on colonic luminal pH, crypt cell proliferation, and colon carcinogenesis were studied in 120 male Sprague-Dawley rats. The animals were divided into five equal groups and fed either a basal fiber free diet or the basal diet supplemented with 10% pectin, cellulose or guar, or 20% oat bran for up to 30 weeks. 1,2-Dimethylhydrazine was given at 20 mg/kg body weight as a weekly s.c. injection for 12 weeks. Food intake and weight gain were similar in all diet groups. At sacrifice, in vivo pH measurements showed that compared to fiber free rats, all fibers significantly acidified large bowel luminal contents (P less than 0.05). In the guar group 62.5% of rats developed colonic tumors compared to 33.4% of the fiber free rats (P less than 0.05). The yield of proximal colonic adenocarcinomas in the oat bran, pectin, and guar groups was increased by 4.5 to 5 times over the fiber free level (P less than 0.05-0.025). Pectin and guar provided the greatest stimulus to cell proliferation. A lower luminal pH was associated with a higher tumor yield and increased epithelial cell proliferation. Thus, acidification of colonic contents by high fiber diets failed to inhibit rat colon carcinogenesis, while the consumption of soluble fibers, such as oat bran, pectin, and guar, was associated with enhancement of proximal colon carcinogenesis.

1,2-Dimethylhydrazine↗

[Immunological demonstration of large quantities of glycogen or of a glycogen-like substance in human embryonic colon cells and in colon carcinomas by means of rabbit antisera raised against a strain of Escherichia coli 013].

Rabbit antisera raised against a strain of E. coli 013, with a strong antiglycogen activity, were tested on human fetal and normal adult colons, on colon carcinomas, and on colon tumor cells in culture (HT29). Only very rare granules were present in adult normal colons when tested with the immunofluorescence method. In faetal colons, in 12 out of 14 carcinomas, and on HT29 cells, the immunofluorescent reactions were similar to those observed in normal liver. The reactions were negative after previous treatment with alpha-amylase. They were inhibited with glycogen, with phenol-alcohol, perchloric, and trichloroacetic extracts from faetal colons, and with a tumor trichloroacetic extract. The extracts precipitated with anti-E. coli 013 antisera. They had a strong inhibiting activity in a radioimmunoassay test with labeled glycogen. The extracts from normal adult colons did not precipitate with the antisera and they had no inhibiting activity in either immunofluorescence and radioimmunoassay tests.

Adult↗

[Development of 1,2-dimethylhydrazine-induced colonic neoplasia in rats and changes in cellular proteins of colonic mucosa].

Development of 1,2-dimethylhydrazine (DMH) induced colonic neoplasia were studied using male Wistar rats given 120 mg DMH per kg s.c. weekly for 5 weeks. During the course of colon carcinogenesis, changes in cellular proteins of colonic mucosa were analysed by two-dimensional gel electrophoresis. Rats were sacrificed just before and at 10, 15 and 20 weeks after the initial DMH treatment together with controls. Incidence and number of colorectal tumors gradually increased. At the 20th week, colon carcinoma was found in every rat. Most tumors (92%) were found in the major flexure and the distal colon and rectum, while only 1% and 7% were found in the cecum and proximal colon, respectively. Histologically, most (92%) were classified as well differentiated or moderately differentiated adenocarcinoma. Eighty-five percent of the tumors were semipedunculated or sessile without depression, and the remainder were sessile with depression. All of the latter were carcinomas with invasion to the submucosa or further. Two-dimensional gel electrophoresis revealed 180 spots in cellular proteins before and after the initial treatment. Three new spots appeared and four spots greatly increased during the course of carcinogenesis, while one spot disappeared. The above results suggest that the appearing and increasing spots may be associated with cancer and that the disappearing spot may be associated with the normal colon.

1,2-Dimethylhydrazine↗

Motor responses to food of the ileum, proximal colon, and distal colon of healthy humans.

Intraluminal pressures and motility indexes were recorded from ileum, ascending colon, rectosigmoid colon, and rectum of 6 healthy men. Three different test meals were eaten at 5-hour intervals. During fasting, migrating motor complexes were identified in the ileum. In ascending colon, irregular, isolated peaks of pressure were common; when bursts of continuous activity occurred, their predominant frequency was 6/min. Regular contractile activity occurred in distal colon at a frequency of 2.5-3.5/min. although 3 subjects also demonstrated rates of 7/min. There were both synchronous and independent contractions of the rectosigmoid and rectum, and no temporal relationship was obvious between motor activities of ascending and distal colons. When the preprandial hour contained no migrating motor complex, all meals increased the ileal motility index by 50%. The motility index of ascending and rectosigmoid colons were enhanced by solid meals, but not by meals containing amino acids. We conclude that motor patterns in the colon vary regionally, both fasting and after food. Results obtained at one site of the large intestine should not be extrapolated to others.

Adult↗

Mucin histochemistry of the colon in relation to the menstrual cycle and its significance in colonic carcinogenesis.

OBJECTIVES: Epidemiological studies indicate an association between reproductive hormonal factors and colon cancers. The present study was undertaken to evaluate the influence of hormonal variations of the menstrual cycle on the mucin histochemistry of normal colonic mucosa and to analyse its significance in relation to colon cancers. METHODS: Fifty three specimens of colon from both 33 reproductive age group females and 20 postmenopausal females were analysed for mucin histochemistry. RESULTS: An increase in the total mucin content was observed in the late progesterone phase with an increase in the sialomucin fraction. CONCLUSIONS: A postulated mechanism for an estrogenic effect include an increased production of bile acids which may act as either promoter or carcinogen in colonic cancers. The cyclical variations of colonic epithelial mucins indicate a sustained estrogen effect is not possible. Hence it is suggested--the regular menstrual cycles play a protective role and some colon cancers may be hormone induced/dependent.

Colon↗

Colon carcinoma glycoproteins carrying alpha 2,6-linked sialic acid reactive with Sambucus nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-Tn antigen.

In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O-acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor-associated sialylated antigens may be constitutively expressed in O-acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma-associated expression of alpha 2,6-linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de-acetylation of normal and transitional colonic mucosa, in contrast to sialyl-Tn antigen, does not result in SNA binding. Further, the alpha 2,6-linked sialic acid recognized by SNA is distinct from that of sialyl-Tn antigen. This is confirmed by Northern blotting detecting transcripts for alpha 2,6 sialyltransferase of N-glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub-lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins.

Acetylation↗

Ultrastructural localization of carcinoembryonic antigen in normal intestine and colon cancer: abnormal distribution of CEA on the surfaces of colon cancer cells.

The distribution of carcinoembryonic antigen (CEA) in normal small intestine, normal colon, and colon cancer of humans was determined immunocytochemically by the peroxidase-labeled antibody method at the light and electron microscopic levels. In the small intestine, CEA was found in protein synthetic organelles, in the mucus, on the microvilli of goblet cells, and on some microvilli of columnar cells adjacent to goblet cells. In the normal colon, CEA was found in protein synthetic organelles of the fully differentiated columnar cells and goblet cells, as well as on the microvilli of the cells. In two well-differentiated colon cancers, the normal preferential surface expression of CEA on the microvilli was maintained, but in six poorly differentiated cancers, CEA was distributed equally over the entire cell surface. We conclude that CEA is a product of goblet cells in the small intestine, columnar and goblet cells in the colon, and colonic cancer cells. CEA on the surfaces of the normal epithelial cells is expressed in a polar manner. This polarity is lacking in undifferentiated neoplastic colon cells, which suggests that failure to establish or maintain the polar expression of normal cell-surface glycoproteins is a characteristic of the neoplastic cells.

Carcinoembryonic Antigen↗

Determinants of differential liver-colonizing potential of variants of the MCA-38 murine colon cancer cell line.

We investigated factors that might contribute to the differing liver tumor colonizing potentials of MCA-38 colonic cancer cell line variants injected into the ileocolic veins of C57Bl/6J mice. Non-colonizing (MCA-38 CD) cells were sensitive to lysis by hepatic natural killer (NK) cells in vitro (51Cr-release assay) and cells with high liver-colonizing potential (MCA-38 LD) were resistant. Following abrogation of NK activity by treatment with anti-asialoGM1, liver-colonizing ability to LD cells but not CD cells was enhanced. MCA-38 CD cells were, however, capable of initial liver colonization after ileocolic vein injection. Differing patterns of membrane sialylation may have contributed to the contrasting hepatic tumorigenicities of LD and CD cells; beta-galactoside alpha 2,6-sialyltransferase mRNA levels and activity were approximately four-fold higher in LD than CD cells and qualitative and quantitative differences existed between their ganglioside profiles. In the MCA-38 model outlined, tumor cell susceptibility or resistance to NK lysis was a relatively unimportant determinant of liver-colonizing potential.

Animals↗

Site distribution of colonic adenomas and carcinomas in relation to colonic flexures.

To minimize the chance of overlooking colonic tumours, 'short segments' of the colon were defined and examined on barium enema films in relation to the distribution of colonic tumours. Films of 299 histologically proven adenomas and carcinomas from 265 consecutive patients were used. No significant differences in distribution were noted between the adenomas and carcinomas. The distance of tumours from distal minor flexures was within seven centimetres in 80% of lesions in the transverse colon, five centimetres in 80% of lesions in the descending colon, and four and six centimetres respectively for 80 and 95% of sigmoid colon lesions. In the cecum plus ascending colon 75% of the lesions were located within seven centimetres from the cecal pole.

Adenoma↗