RESUMEN
Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by generating cytotoxic and oxidative stress. Recently, we found that this surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a changed surface to crypt signaling. It is unknown whether this changed signaling is caused by cytotoxic stress and/or oxidative stress, as these processes were never studied separately. The aim of this study was to determine the possible differential effects of dietary heme on these luminal stressors and their impact on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding. Mice received a purified, humanized, control diet or the diet supplemented with 0.2 µmol heme/g. Oxidative and cytotoxic stress were measured in fecal water. Proliferation was determined by Ki67-immunohistochemistry and mucosal responses by whole-genome transcriptomics. After heme ingestion, there was an acute increase in reactive oxygen species (ROS) leading to increased levels of lipid peroxidation products. Mucosal gene expression showed an acute antioxidant response, but no change in cell turnover. After day 4, cytotoxicity of the colonic contents was increased and this coincided with differential signaling and hyperproliferation, indicating that cytotoxicity was the causal factor. Simultaneously, several oncogenes were activated, whereas the tumor suppressor p53 was inhibited. In conclusion, luminal cytotoxicity, but not ROS, caused differential surface to crypt signaling resulting in mucosal hyperproliferation and the differential expression of oncogenes and tumor suppressor genes.
Asunto(s)
Proliferación Celular , Colon/efectos de los fármacos , Suplementos Dietéticos , Regulación Neoplásica de la Expresión Génica , Hemo/administración & dosificación , Estrés Oxidativo , Animales , Colon/química , Colon/patología , Heces/química , Hemo/farmacología , Inmunohistoquímica , Mucosa Intestinal/química , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Peroxidación de Lípido , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/química , Factores de Tiempo , TranscriptomaRESUMEN
The Western diet, rich in fat and red meat, predisposes for inflammatory bowel disease (IBD); however, little is known about mechanisms involved. Red meat contains high levels of heme, a well-known inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1). Pharmacological induction of HO-1 ameliorates experimental colitis. We analyzed the effect of a westernized high-fat (HF) diet supplemented with heme on intestinal HO-1 expression and dextran sulfate sodium (DSS)-induced colitis. Mice were fed chow or HF diets for 2 weeks. In the second week, the HF diet was supplemented with or without 0.5 µmol/g heme. Subsequently, the 3 diet groups were given drinking water with or without 4% DSS to induce colitis. Significant body weight reduction was first observed after 4 days in the chow/DSS mice (-5±3%), whereas this was evident already after 2 days (-6±2%) in HF/DSS mice, showing increased weight loss compared to chow/DSS mice in the following days. Heme supplementation further aggravated DSS-induced weight loss in HF mice (-18±4% vs. -7±5% for HF+heme/DSS vs. HF/DSS, P<.01). Heme increased HO-1 expression in the colon epithelium but decreased villin messenger RNA levels, indicating epithelial damage. In contrast, heme did not affect DSS-induced colon shortening and histological scores of epithelial damage and inflammation. A westernized diet accelerates DSS-induced weight loss in mice, which is further aggravated by heme, despite the induction of HO-1 in the colon epithelium. Our data warrant a detailed analysis of the association of (red) meat-containing diets and the development of IBD.
Asunto(s)
Colitis/patología , Sulfato de Dextran , Dieta Alta en Grasa , Hemo/administración & dosificación , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colon/metabolismo , Colon/patología , Suplementos Dietéticos , Femenino , Hemo/farmacología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Pérdida de PesoRESUMEN
An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microbial fermentation. Therefore, we investigated whether dietary phosphate is a co-player in the effect of Ca on permeability. Rats were fed a humanised low-Ca diet, or a similar diet supplemented with Ca and containing either high, medium or low phosphate concentrations. Chromium-EDTA was added as an inert dietary intestinal permeability marker. After dietary adaptation, short-chain fructo-oligosaccharides (scFOS) were added to all diets to stimulate fermentation, acidify the colonic contents and induce an increase in permeability. Dietary Ca prevented the scFOS-induced increase in intestinal permeability in rats fed medium- and high-phosphate diets but not in those fed the low-phosphate diet. This was associated with higher faecal water cytotoxicity and higher caecal lactate levels in the latter group. Moreover, food intake and body weight during scFOS supplementation were adversely affected by the low-phosphate diet. Importantly, luminal buffering capacity was higher in rats fed the medium- and high-phosphate diets compared with those fed the low-phosphate diet. The protective effect of dietary Ca on intestinal permeability is impaired if dietary phosphate is low. This is associated with a calcium phosphate-induced increase in luminal buffering capacity. Dragging phosphate into the colon and thereby increasing the colonic phosphate concentration is at least part of the mechanism behind the protective effect of Ca on intestinal permeability.
Asunto(s)
Calcio de la Dieta/administración & dosificación , Colon/efectos de los fármacos , Colon/fisiología , Animales , Tampones (Química) , Fosfatos de Calcio/metabolismo , Ciego/efectos de los fármacos , Ciego/metabolismo , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/metabolismo , Fermentación , Ácido Láctico/metabolismo , Masculino , Oligosacáridos/administración & dosificación , Oligosacáridos/metabolismo , Permeabilidad/efectos de los fármacos , Fosfatos/administración & dosificación , Fosfatos/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: Colon cancer is a leading cause of cancer deaths in Western countries and is associated with diets high in red meat. Haem, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents and damages the colon surface epithelium. Compensatory hyperproliferation leads to epithelial hyperplasia which increases the risk of colon cancer. The aim of this study was to identify molecules signalling from the surface epithelium to the crypt to initiate hyperproliferation upon stress induced by haem. METHODS: C57Bl6/J mice (n=9/group) received a 'westernised' control diet (40 en% fat) with or without 0.5 µmol/g haem for 14 days. Colon mucosa was used to quantify cell proliferation and for microarray transcriptome analysis. Gene expression profiles of surface and crypt cells were compared using laser capture microdissection. Protein levels of potential signalling molecules were quantified. RESULTS: Haem-fed mice showed epithelial hyperproliferation and decreased apoptosis, resulting in hyperplasia. Microarray analysis of the colon mucosa showed 3710 differentially expressed genes (false discovery rate (q) <0.01), with many involved in the cell cycle. Expression levels of haem- and stress-related genes showed that haem affected surface cells but did not directly affect crypt cells. Injured surface cells should therefore signal to crypt cells to induce compensatory hyperproliferation. Haem downregulated the inhibitors of proliferation, Wnt inhibitory factor 1, Indian Hedgehog and bone morphogenetic protein 2. Interleukin-15 was also downregulated. Haem upregulated amphiregulin, epiregulin and cyclo-oxygenase-2 mRNA in surface cells. Their protein/metabolite levels were, however, not increased as haem induced surface-specific inhibition of translation by increasing 4E-BP1. CONCLUSIONS: Haem induces colonic hyperproliferation and hyperplasia by inhibiting the surface to crypt signalling of feedback inhibitors of proliferation.
Asunto(s)
Colon/citología , Células Epiteliales/efectos de los fármacos , Hemo/farmacología , Mucosa Intestinal/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Colon/metabolismo , Neoplasias del Colon/etiología , Suplementos Dietéticos , Regulación hacia Abajo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Retroalimentación Fisiológica , Expresión Génica , Perfilación de la Expresión Génica , Captura por Microdisección con Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , TranscriptomaRESUMEN
BACKGROUND: Oxidative stress is presumed to play an important role in inflammatory bowel disease (IBD). Accordingly, antioxidant supplementation might be protective. Dietary calcium inhibited colitis development in HLA-B27 transgenic rats, an animal model mimicking IBD. As antioxidants might act at mucosa level and calcium predominantly in the gut lumen, we hypothesize that the combination has additive protective effects on colitis development. METHODS: HLA-B27 rats were fed a control diet or the same diet supplemented with the antioxidants glutathione, vitamin C, and vitamin E, or supplemented with both antioxidants and calcium. Oxidative stress in colonic mucosa, colonic inflammation, intestinal permeability, and diarrhea were quantified. RESULTS: Intestinal permeability, diarrhea, myeloperoxidase, and interleukin-1ß levels were significantly lower in rats fed both antioxidants and calcium compared to rats supplemented with antioxidants only. No beneficial effects were observed in rats fed the diet supplemented with antioxidants only. Strikingly, despite extremely low colonic mucosal glutathione levels in HLA-B27 rats, there was no oxidative stress-related damage. Subsequent analyses showed no defect in expression of glutathione synthesis genes. Additional experiments, comparing young and older HLA-B27 rats, showed that glutathione levels and also reactive oxygen species production decreased with progression of intestinal inflammation. CONCLUSIONS: Antioxidant supplementation was ineffective in HLA-B27 rats despite low mucosal glutathione levels, because colitis development did not coincide with oxidative stress in this model. This indicates that the neutrophilic respiratory burst, and thus innate immune defense, is compromised in HLA-B27 rats. As supplementation with both calcium and antioxidants attenuated colitis development, we speculate that this protective effect is attributed to calcium only.
Asunto(s)
Antioxidantes/administración & dosificación , Calcio de la Dieta/administración & dosificación , Colitis/patología , Suplementos Dietéticos , Glutatión/metabolismo , Antígeno HLA-B27/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colitis/tratamiento farmacológico , Colitis/metabolismo , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , ARN Mensajero/genética , Ratas , Ratas Transgénicas , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
There is increased interest in the potential protective role of dietary Ca in the development of metabolic disorders related to the metabolic syndrome. Ca-induced intestinal precipitation of fatty acids and bile acids as well as systemic metabolic effects of Ca on adipose tissue is proposed to play a causal role. In this experiment, we have studied all these aspects to validate the suggested protective effect of Ca supplementation, independent of other dietary changes, on the development of diet-induced obesity and insulin resistance. In our diet intervention study, C57BL/6J mice were fed high-fat diets differing in Ca concentrations (50 v. 150 mmol/kg). Faecal excretion analyses showed an elevated precipitation of intestinal fatty acids (2·3-fold; P < 0·01) and bile acids (2-fold; P < 0·01) on the high-Ca diet. However, this only led to a slight reduction in fat absorption (from 98 to 95 %; P < 0·01), mainly in the distal small intestine as indicated by gene expression changes. We found no effect on body-weight gain. Lipolysis and lipogenesis-related parameters in adipose tissue also showed no significant changes on the high-Ca diet, indicating no systemic effects of dietary Ca on adiposity. Furthermore, early gene expression changes of intestinal signalling molecules predicted no protective effect of dietary Ca on the development of insulin resistance, which was confirmed by equal values for insulin sensitivity on both diets. Taken together, our data do not support the proposed protective effect of dietary Ca on the development of obesity and/or insulin resistance, despite a significant increase in faecal excretion of fatty acids and bile acids.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Calcio de la Dieta/farmacología , Grasas de la Dieta/metabolismo , Ácidos Grasos/metabolismo , Resistencia a la Insulina , Intestino Delgado/efectos de los fármacos , Obesidad/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Grasas de la Dieta/efectos adversos , Suplementos Dietéticos , Heces/química , Expresión Génica/efectos de los fármacos , Absorción Intestinal , Intestino Delgado/metabolismo , Lipogénesis/efectos de los fármacos , Lipólisis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/inducido químicamente , Oligoelementos/farmacologíaRESUMEN
Perturbation of the intestinal microbiota by antibiotics predisposes the host to food-borne pathogens like Salmonella. The effects of antibiotic treatment on intestinal permeability during infection and the efficacy of dietary components to improve resistance to infection have not been studied. Therefore, we investigated the effect of clindamycin on intestinal barrier function in Salmonella-infected rats. We also studied the ability of dietary calcium and tannic acid to protect against infection and concomitant diarrhea and we assessed intestinal barrier function. Rats were fed a purified control diet including the permeability marker chromium EDTA (CrEDTA) (2 g/kg) or the same diet supplemented with calcium (4.8 g/kg) or tannic acid (3.75 g/kg). After adaptation, rats were orally treated with clindamycin for 4 d followed by oral infection with Salmonella enteritidis. Two additional control groups were not treated with antibiotics and received either saline or Salmonella. Urine and feces were collected to quantify intestinal permeability, diarrhea, cytotoxicity of fecal water, and Salmonella excretion. In addition, Salmonella translocation was determined. Diarrhea, CrEDTA excretion, and cytotoxicity of fecal water were higher in the clindamycin-treated infected rats than in the non-clindamycin-treated infected control group. Intestinal barrier function was less in the Salmonella-infected rats pretreated with antibiotics compared with the non-clindamycin- treated rats. Both calcium and tannic acid reduced infection-associated diarrhea and inhibited the adverse intestinal permeability changes but did not decrease Salmonella colonization and translocation. Our results indicate that calcium protects against intestinal changes due to Salmonella infection by reducing luminal cytotoxicity, whereas tannic acid offers protection by improving the mucosal resistance.
Asunto(s)
Antibacterianos/farmacología , Calcio de la Dieta/administración & dosificación , Mucosa Intestinal/efectos de los fármacos , Infecciones por Salmonella/patología , Taninos/administración & dosificación , Animales , Mucosa Intestinal/microbiología , Masculino , Ratas , Ratas WistarRESUMEN
An increased intestinal permeability is associated with several diseases. Nutrition can influence gut permeability. Previously, we showed that dietary Ca decreases whereas dietary short-chain fructo-oligosaccharides (scFOS) increase intestinal permeability in rats. However, it is unknown how and where in the gastrointestinal tract Ca and scFOS exert their effects. Rats were fed a Western low-Ca control diet, or a similar diet supplemented with either Ca or scFOS. Lactulose plus mannitol and Cr-EDTA were added to the diets to quantify small and total gastrointestinal permeability, respectively. Additionally, colonic tissue was mounted in Ussing chambers and exposed to faecal water of these rats. Dietary Ca immediately decreased urinary Cr-EDTA excretion by 24 % in Ca-fed rats compared with control rats. Dietary scFOS increased total Cr-EDTA permeability gradually with time, likely reflecting relatively slow gut microbiota adaptations, which finally resulted in a 30 % increase. The lactulose:mannitol ratio was 15 % higher for Ca-fed rats and 16 % lower for scFOS-fed rats compared with control rats. However, no dietary effect was present on individual urinary lactulose and mannitol excretion. The faecal waters did not influence colonic permeability in Ussing chambers. In conclusion, despite effects on the lactulose:mannitol ratio, individual lactulose values did not alter, indicating that diet did not influence small-intestinal permeability. Therefore, both nutrients affect permeability only in the colon: Ca decreases, while scFOS increase colonic permeability. As faecal water did not influence permeability in Ussing chambers, probably modulation of mucins and/or microbiota is important for the in vivo effects of dietary Ca and scFOS.
Asunto(s)
Calcio de la Dieta/farmacología , Colon/efectos de los fármacos , Oligosacáridos/química , Oligosacáridos/farmacología , Animales , Colon/metabolismo , Dieta , Heces/química , Masculino , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Agua/análisisRESUMEN
We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.
Asunto(s)
Calcio de la Dieta/uso terapéutico , Calcio/uso terapéutico , Colitis/tratamiento farmacológico , Diarrea/tratamiento farmacológico , Matriz Extracelular/efectos de los fármacos , Absorción Intestinal/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Calcio/farmacología , Calcio de la Dieta/farmacología , Colitis/genética , Colitis/metabolismo , Colon/efectos de los fármacos , Colon/inmunología , Colon/metabolismo , Diarrea/metabolismo , Suplementos Dietéticos , Modelos Animales de Enfermedad , Ácido Edético/administración & dosificación , Ácido Edético/orina , Heces , Femenino , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica/efectos de los fármacos , Antígeno HLA-B27/genética , Interleucina-1beta/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Permeabilidad/efectos de los fármacos , Procolágeno/genética , Procolágeno/metabolismo , Ratas , Ratas TransgénicasRESUMEN
BACKGROUND: Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.Rats were fed a control diet or the same diet supplemented with buthionine sulfoximine (BSO; glutathione depletion) or cystine (glutathione maintenance). Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability. At day 4 after oral gavage with Salmonella enteritidis (or saline for non-infected controls), Salmonella translocation was determined by culturing extra-intestinal organs. Liver and ileal mucosa were collected for analyses of glutathione, inflammation markers and oxidative damage. Faeces was collected to quantify diarrhoea. RESULTS: Glutathione depletion aggravated ileal inflammation after infection as indicated by increased levels of mucosal myeloperoxidase and interleukin-1beta. Remarkably, intestinal permeability and Salmonella translocation were not increased. Cystine supplementation maintained glutathione in the intestinal mucosa but inflammation and oxidative damage were not diminished. Nevertheless, cystine reduced intestinal permeability and Salmonella translocation. CONCLUSION: Despite increased infection-induced mucosal inflammation upon glutathione depletion, this tripeptide does not play a role in intestinal permeability, bacterial translocation and diarrhoea. On the other hand, cystine enhances gut barrier function by a mechanism unlikely to be related to glutathione.
Asunto(s)
Traslocación Bacteriana/fisiología , Glutatión/fisiología , Mucosa Intestinal/fisiología , Salmonelosis Animal/fisiopatología , Animales , Traslocación Bacteriana/efectos de los fármacos , Butionina Sulfoximina/farmacología , Cistina/administración & dosificación , Cistina/farmacología , Diarrea/etiología , Diarrea/fisiopatología , Susceptibilidad a Enfermedades , Glutatión/antagonistas & inhibidores , Ileítis/fisiopatología , Interleucina-1beta/análisis , Lipopolisacáridos/toxicidad , Hígado/metabolismo , Masculino , Óxido Nítrico/metabolismo , Estrés Oxidativo , Peroxidasa/análisis , Ratas , Ratas Wistar , Salmonelosis Animal/complicaciones , Salmonelosis Animal/microbiología , Salmonella enteritidis/fisiología , Organismos Libres de Patógenos EspecíficosRESUMEN
Transporters present in the epithelium of the small intestine determine the efficiency by which dietary and biliary cholesterol are taken up into the body and thus control whole-body cholesterol balance. Niemann-Pick C1 Like Protein 1 (Npc1l1) transports cholesterol into the enterocyte, whereas ATP-binding cassette transporters Abca1 and Abcg5/Abcg8 are presumed to be involved in cholesterol efflux from the enterocyte toward plasma HDL and back into the intestinal lumen, respectively. Abca1, Abcg5, and Abcg8 are well-established liver X receptor (LXR) target genes. We examined the effects of a high-fat diet on expression and function of cholesterol transporters in the small intestine in mice. Npc1l1, Abca1, Abcg5, and Abcg8 were all downregulated after 2, 4, and 8 wk on a cholesterol-free, high-fat diet. The high-fat diet did not affect biliary cholesterol secretion but diminished fractional cholesterol absorption from 61 to 42% (P < 0.05). In an acute experiment in which triacylglycerols of unsaturated fatty acids were given by gavage, we found that this downregulation occurs within a 6-h time frame. Studies in LXRalpha-null mice, confirmed by in vitro data, showed that fatty acid-induced downregulation of cholesterol transporters is LXRalpha independent and associated with a posttranslational increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity that reflects induction of cholesterol biosynthesis as well as with a doubling of neutral fecal sterol loss. This study highlights the induction of adaptive changes in small intestinal cholesterol metabolism during exposure to dietary fat.
Asunto(s)
Colesterol/metabolismo , Grasas de la Dieta/farmacología , Intestino Delgado/metabolismo , Proteínas de Transporte de Membrana/genética , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/análisis , Colesterol/sangre , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Grasas de la Dieta/administración & dosificación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Heces/química , Regulación de la Expresión Génica/efectos de los fármacos , Hidroxicolesteroles/farmacología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Lipoproteínas/genética , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triglicéridos/farmacologíaRESUMEN
In contrast to most expectations, we showed previously that dietary fructooligosaccharides (FOS) stimulate intestinal colonization and translocation of invasive Salmonella enteritidis in rats. Even before infection, FOS increased the cytotoxicity of fecal water, mucin excretion, and intestinal permeability. In the present study, we tested whether FOS has these effects in humans. A double-blind, placebo-controlled, crossover study of 2 x 2 wk, with a washout period of 2 wk, was performed with 34 healthy men. Each day, subjects consumed lemonade containing either 20 g FOS or placebo and the intestinal permeability marker chromium EDTA (CrEDTA). On the last 2 d of each supplement period, subjects scored their gastrointestinal complaints on a visual analog scale and collected feces and urine for 24 h. Fecal lactic acid was measured using a colorimetric enzymatic kit. The cytotoxicity of fecal water was determined with an in vitro bioassay, fecal mucins were quantified fluorimetrically, and intestinal permeability was determined by measuring urinary CrEDTA excretion. In agreement with our animal studies, FOS fermentation increased fecal wet weight, bifidobacteria, lactobacilli, and lactic acid. Consumption of FOS increased flatulence and intestinal bloating. In addition, FOS consumption doubled fecal mucin excretion, indicating mucosal irritation. However, FOS did not affect the cytotoxicity of fecal water and intestinal permeability. The FOS-induced increase in mucin excretion in our human study suggests mucosal irritation in humans, but the overall effects are more moderate than those in rats.
Asunto(s)
Intestinos/efectos de los fármacos , Mucinas/efectos de los fármacos , Oligosacáridos/efectos adversos , Adulto , Estudios Cruzados , Dieta , Método Doble Ciego , Heces/química , Fermentación/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/microbiología , Masculino , Persona de Mediana Edad , Mucinas/aislamiento & purificación , Oligosacáridos/administración & dosificaciónRESUMEN
Diets high in red meat and low in green vegetables are associated with an increased risk of colon cancer. In rats, dietary heme, mimicking red meat, increases colonic cytotoxicity and proliferation of the colonocytes, whereas addition of chlorophyll from green vegetables inhibits these heme-induced effects. Chlorophyllin is a water-soluble hydrolysis product of chlorophyll that inhibits the toxicity of many planar aromatic compounds. The present study investigated whether chlorophyllins could inhibit the heme-induced luminal cytotoxicity and colonic hyperproliferation as natural chlorophyll does. Rats were fed a purified control diet, the control diet supplemented with heme, or a heme diet with 1.2 mmol/kg diet of chlorophyllin, copper chlorophyllin, or natural chlorophyll for 14 d (n = 8/group). The cytotoxicity of fecal water was determined with an erythrocyte 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 feces using quantitative PCR analysis. Heme caused a >50-fold increase in the cytotoxicity of the fecal water, a nearly 100% increase in proliferation, and almost total inhibition of exfoliation of the colonocytes. Furthermore, the addition of heme increased TBARS in fecal water. Chlorophyll, but not the chlorophyllins, completely prevented these heme-induced effects. In conclusion, inhibition of the heme-induced colonic cytotoxicity and epithelial cell turnover is specific for natural chlorophyll and cannot be mimicked by water-soluble chlorophyllins.
Asunto(s)
Anticarcinógenos/farmacología , División Celular/efectos de los fármacos , Clorofila/farmacología , Clorofilidas/farmacología , Colon/fisiopatología , Hemo/metabolismo , Verduras , Animales , Colon/efectos de los fármacos , Colon/fisiología , Heces/química , Peroxidación de Lípido , Masculino , Modelos Animales , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
We showed previously that fructooligosaccharides (FOS) decrease the resistance to salmonella infection in rats. However, the mechanism responsible for this effect is unclear. Therefore, we examined whether dietary FOS affects intestinal permeability before and after infection with Salmonella enterica serovar Enteritidis. Male Wistar rats were fed restricted quantities of a purified diet that mimicked the composition of a Western human diet. The diet was supplemented with 60 g/kg cellulose (control) or 60 g/kg FOS and with 4 mmol/kg of the intestinal permeability marker chromium EDTA (CrEDTA) (n = 8 or 10). After an adaptation period of 2 wk, rats were orally infected with 10(8) colony-forming units (cfu) of S. enteritidis. Mucin concentrations in intestinal contents and mucosa were measured fluorimetrically, as markers of mucosal irritation. Intestinal permeability was determined by measuring urinary CrEDTA excretion. Translocation of salmonella was quantified by analysis of urinary nitric oxide metabolites with time. Before infection, FOS increased mucosal lactobacilli and enterobacteria in cecum and colon, but not in the ileum. However, FOS increased cytotoxicity of fecal water and intestinal permeability. Moreover, FOS increased fecal mucin excretion and mucin concentrations in cecal and colonic contents, and in cecal mucosa before infection. After infection, mucin excretion and intestinal permeability in the FOS groups increased even further in contrast to the control group. In addition, FOS increased translocation of salmonella to extraintestinal sites. Thus, FOS impairs the intestinal barrier in rats, as indicated by higher intestinal permeability. Whether these results can be extrapolated to humans requires further investigation.
Asunto(s)
Absorción Intestinal/fisiología , Mucosa Intestinal/fisiología , Oligosacáridos/farmacología , Animales , Dieta , Heces/química , Heces/microbiología , Concentración de Iones de Hidrógeno , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Intestinos/fisiología , Masculino , Oligosacáridos/administración & dosificación , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
División Celular/efectos de los fármacos , Clorofila/farmacología , Neoplasias del Colon/patología , Hemo/metabolismo , Carne/efectos adversos , Animales , División Celular/fisiología , Células Cultivadas , Clorofila/química , Colon/efectos de los fármacos , Colon/patología , Neoplasias del Colon/dietoterapia , Neoplasias del Colon/etiología , Dieta/efectos adversos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Heces/química , Masculino , Ratas , Ratas Wistar , Spinacia oleracea/químicaRESUMEN
Red meat intake is associated with colon cancer risk. Puzzlingly, meat does not promote carcinogenesis in rat studies. However, we demonstrated previously that dietary heme promotes aberrant crypt foci (ACF) formation in rats given a low-calcium diet. Here, we tested the hypothesis that heme-rich meats promote colon carcinogenesis in rats treated with azoxymethane and fed low-calcium diets (0.8 g/kg). Three meat-based diets were formulated to contain varying concentrations of heme by the addition of raw chicken (low heme), beef (medium heme), or black pudding (blood sausage; high heme). The no-heme control diet was supplemented with ferric citrate and the heme control diet with hemoglobin to match iron and heme concentrations in the beef diet, respectively. After 100 d, colons were scored for ACF and mucin-depleted foci (MDF). Fecal water was assayed for lipoperoxides and cytotoxicity. Only diets with heme promoted the formation of MDF, but all meat diets promoted ACF formation. The number of MDF/colon was 0.55 +/- 0.68 in controls, but 1.2 +/- 0.6 (P = 0.13), 1.9 +/- 1.4 (P < 0.01), and 3.0 +/- 1.2 (P < 0.001) in chicken-, beef-, and black pudding-fed rats. MDF promotion by the high-heme black pudding diet was greater than that by the medium-heme beef diet. The number of ACF/colon was 72 +/- 16 in controls, but 91 +/- 18, 100 +/- 13, and 103 +/- 14 in chicken-, beef-, and black pudding-fed rats (all P < 0.001). ACF and MDF did not differ between rats fed the beef diet and those fed the heme control diet. MDF promotion was correlated with high fecal water lipoperoxides and cytotoxicity (r = 0.65, P < 0.01). This is the first study to show the promotion of experimental carcinogenesis by dietary meat and the association with heme intake.
Asunto(s)
Azoximetano/toxicidad , Carcinógenos/toxicidad , Neoplasias del Colon/inducido químicamente , Dieta , Hemo/administración & dosificación , Carne , Mucinas/deficiencia , Animales , Bovinos , Pollos , Femenino , Peroxidación de Lípido/efectos de los fármacos , Mucinas/efectos de los fármacos , Ratas , Ratas Endogámicas F344RESUMEN
BACKGROUND & AIMS: In several rat infection experiments, we have shown that dietary calcium inhibits intestinal colonization and translocation of invasive salmonella. The aim of the present study was to find out whether calcium is also protective against enterotoxigenic Escherichia coli (ETEC) infection. This was first tested in our rat model and subsequently verified in a human infection study. METHODS: Rats were fed a purified diet with either a low or a high amount of calcium phosphate and orally infected with ETEC. In addition, a parallel, double-blind, placebo-controlled intervention study of 3 weeks was performed with 32 healthy men. Subjects largely maintained their habitual diet and consumed either regular milk products (calcium supply, 1100 mg/day) or placebo milk products (calcium supply, 60 mg/day). On day 10, subjects ingested a live but attenuated ETEC strain (strain E1392/75-2A), able to induce mild although short-lived symptoms. Primary outcomes studied were infection-induced diarrhea (total fecal output and relative fecal dry weight) and fecal mucin excretion. RESULTS: In humans, ETEC induced diarrhea in both groups, in that total fecal output doubled and mean relative fecal dry weight dropped from 25% to 20%. Additionally, fecal mucin excretion was increased in both groups. All these fecal parameters were completely normalized in the calcium group on the second infection day, in contrast to the placebo group, which recovered on the third infection day. Likewise, supplemental calcium inhibited ETEC colonization and diarrhea in rats. CONCLUSIONS: Calcium in milk products improves human resistance to ETEC infection as it inhibits infectious diarrhea.
Asunto(s)
Calcio de la Dieta/administración & dosificación , Diarrea/prevención & control , Infecciones por Escherichia coli/prevención & control , Adulto , Animales , Método Doble Ciego , Heces/microbiología , Humanos , Masculino , Persona de Mediana Edad , Mucinas/metabolismo , Ratas , Ratas WistarRESUMEN
High intake of red meat, but not of white meat, is associated with an increased risk of colon cancer. However, red meat does not promote cancer in rodents. Haemin, added to low-calcium diets, increases colonic proliferation, and haemoglobin, added to high-fat diets, increases the colon tumour incidence in rats, an effect possibly due to peroxyl radicals. We thus speculated that haem might be the promoting agent in meat, and that prevention strategies could use calcium and antioxidants. These hypotheses were tested in rats at the aberrant crypt foci (ACF) stage at 100 days. F344 rats (n = 124) were given an injection of azoxymethane and were then randomized to 11 groups fed with low-calcium (20 micro mol/g) AIN76-based diets, containing 5% safflower oil. Haemin (0.25, 0.5 and 1.5 micro mol/g) or haemoglobin (1.5 and 3 micro mol haem/g) was added to five experimental diets, compared with a control diet without haem. Three other high-haemin diets (1.5 micro mol/g) were supplemented with calcium (250 micro mol/g), antioxidant butylated hydroxyanisole and rutin (0.05% each), and olive oil, which replaced safflower oil. Faecal water was assayed for lipid peroxidation by thiobarbituric acid reactive substances (TBARs) test, and for cytolytic activity. Haemin strikingly increased the ACF size, dose-dependently, from 2.6 to 11.4 crypts/ACF (all P < 0.001). The high-haemin diet also increased the number of ACF per colon (P < 0.001). Promotion was associated with increased faecal water TBARs and cytotoxicity. Calcium, olive oil and antioxidants each inhibited the haemin-induced ACF promotion, and normalized the faecal TBARs and cytotoxicity. The haemoglobin diets increased the number of ACF and faecal TBARs, but not the ACF size or the faecal cytotoxicity. In conclusion, dietary haemin is the most potent known ACF promoter. Haemoglobin is also a potent promoter of colorectal carcinogenesis. The results suggest that myoglobin in red meat could promote colon cancer. Diets high in calcium, or in oxidation-resistant fats, may prevent the possible cancer-promoting effect of red meat.
Asunto(s)
Colon/efectos de los fármacos , Colon/patología , Neoplasias Colorrectales/etiología , Hemina/efectos adversos , Hemoglobinas/efectos adversos , Carne/efectos adversos , Animales , Antioxidantes/farmacología , Azoximetano/toxicidad , Hidroxianisol Butilado/farmacología , Calcio/metabolismo , Dieta , Relación Dosis-Respuesta a Droga , Heces/química , Femenino , Hemo/análisis , Peroxidación de Lípido/efectos de los fármacos , Aceite de Oliva , Aceites de Plantas/farmacología , Ratas , Ratas Endogámicas F344 , Rutina/farmacología , Aceite de Cártamo/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico , Aumento de PesoRESUMEN
Patients with a history of sporadic adenomas have increased epithelial cell proliferative activity, an intermediate risk marker for colorectal cancer. Reduction of proliferation by dietary intervention may reflect a decreased colorectal cancer risk. To evaluate whether calcium or resistant starch could reduce proliferative activity throughout the colon, we performed a randomized controlled trial in 111 sporadic adenoma patients. Patients received two placebos, 1 g of calcium + placebo, or 30 g of amylomaize (19 g of resistant starch) + placebo. After 2 mo, biopsies were collected from the cecum, transverse and sigmoid colon, and rectum during colonoscopy. Epithelial cell proliferation was determined by dividing the number of 5-bromo-2-deoxyuridine-labeled nuclei by the total number of nuclei x 100 (labeling index, LI). LI of luminal, mid, and basal compartments was determined. Twenty-five patients dropped out. In the remaining 86 patients (28 treated with placebo, 30 with calcium + placebo, and 28 with resistant starch + placebo), no difference was observed in total LI, the LI of the three compartments, or the crypt length in the four areas of the colorectum. Colonic epithelial cell proliferative activity throughout the colon of sporadic adenoma patients is not affected by supplementation with 1 g of calcium or 19 g of resistant starch.
Asunto(s)
Adenoma/dietoterapia , Calcio de la Dieta/administración & dosificación , Colon/efectos de los fármacos , Colon/patología , Neoplasias Colorrectales/dietoterapia , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Almidón/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , División Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Most Gram-positive bacteria are susceptible to the bactericidal action of fatty acids and bile acids. Because dietary calcium phosphate (CaP(i)) lowers the intestinal concentration of these antimicrobial agents, high CaP(i) intake may enhance intestinal colonization of Gram-positive pathogens and the subsequent pathogenesis. In this study, we tested this hypothesis in a rat model using Listeria monocytogenes. Rats were fed diets containing low (20 micromol/g diet) or high (160 micromol/g diet) amounts of CaP(i). Dietary fat was provided as corn oil or milk fat. Rats were orally inoculated with L. monocytogenes. When rats consumed diets containing corn oil, high CaP(i) intake indeed stimulated colonization of L. monocytogenes and increased L. monocytogenes translocation and diarrhea. In addition, supplemental CaP(i) enhanced ex vivo growth of L. monocytogenes in fecal extracts of rats fed corn oil diets, suggesting that high CaP(i) intake decreased a luminal inhibitory factor. The concentrations of bile salts and fatty acids, which were highly listericidal in vitro, were indeed considerably decreased in fecal water of rats in the high calcium corn oil group. Surprisingly, dietary CaP(i) did not affect colonization and translocation of L. monocytogenes in rats fed the milk fat diet, nor did CaP(i) enhance ex vivo growth in fecal extracts. This absence of Listeria stimulation was associated with a lack of effect of dietary CaP(i) on fecal soluble fatty acids. In addition, residual soluble bile salts were higher in rats fed the high CaP(i) milk fat diet compared with the high CaP(i) corn oil diet. These results suggest that the stimulating effect of CaP(i) on L. monocytogenes infection depends on the type of dietary fat consumed.