Dietary heme induces acute oxidative stress, but delayed cytotoxicity and compensatory hyperproliferation in mouse colon.

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.

Dietary galacto-oligosaccharides and calcium: effects on energy intake, fat-pad weight and satiety-related, gastrointestinal hormones in rats.

Galacto-oligosaccharides (GOS) are carbohydrates that are fermented by colonic microbiota. The present study examined effects of a 3-week dietary enrichment with 6 % (w/w) GOS on parameters of energy balance in forty-three male Wistar rats. GOS was tested with two doses of calcium phosphate (30 and 100 mmol/kg), known to differently affect colonic fermentation. After 17 d, isoenergetic test meals were presented and plasma responses of ghrelin, glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were measured. On day 21 (study termination) epididymal fat pads and caecum were weighed. Additionally, gastrointestinal mucosal samples and proximal colonic contents were analysed for gene expression (ghrelin, proglucagon and PYY) and fermentation metabolites (SCFA and lactate), respectively. GOS reduced energy intake most prominently during the first week, without provoking compensatory overeating later on (average intake reduction: 14 %). The GOS-fed rats showed increased caecal and reduced fat-pad weight and increased gene expression of the satiety-related peptides, PYY (1.7-fold) and proglucagon (3.5-fold). Pre-meal baseline and post-meal plasma levels of PYY, but not of ghrelin or GLP-1, were higher in GOS-fed rats than in control rats. Ca enrichment resulted in higher energy intake (average 4.5 %). GOS diets increased lactic acid levels and slightly reduced butyric acid in proximal colonic contents. Ca abolished the GOS-related elevation of lactic acid, while increasing propionic acid levels, but did not inhibit GOS-related effects on energy intake, fat-pad weight or gene expression. These results indicate that dietary GOS stimulate a number of physiological mechanisms that can reduce energy intake, regardless of the calcium phosphate content of the diet.

Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice.

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b(-/-)) mice and wild-type (WT) mice were orally infected with gram-negative Salmonella enteritidis or gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b(-/-) mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b(-/-) mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the gram-negative bacterium S. enteritidis in mice but not against the gram-positive bacterium L. monocytogenes.

The protective effect of supplemental calcium on colonic permeability depends on a calcium phosphate-induced increase in luminal buffering capacity.

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.

Efficacy of various dietary calcium salts to improve intestinal resistance to Salmonella infection in rats.

Previous animal and human studies have shown protective effects of Ca on the resistance to enteropathogenic infections. Most interventions were performed with calcium phosphate and little is known about the protective effect of other dietary sources of Ca. Therefore, we investigated the efficacy of several Ca salts to enhance intestinal resistance to Salmonella enteritidis infection. Rats (n 7-8 per group) were fed a high-fat, Western human-style, purified diet with a low Ca content (20 mmol calcium phosphate/kg; negative control group) or the same diet supplemented with either (extra) calcium phosphate, milk Ca, calcium chloride or calcium carbonate (total of 100 mmol Ca supplement/kg). Diets contained Cr-EDTA for assessment of incremental changes in intestinal permeability. After an adaptation period of 2 weeks, animals were orally infected with S. enteritidis to mimic a human-relevant foodborne infection. Ca supplement-induced changes on faecal lactobacilli and enterobacteria were studied before infection. Changes in intestinal permeability were determined by measuring urinary Cr with time. Persistence of Salmonella was determined by studying faecal excretion of this pathogen in time. Overall, all Ca salts increased resistance towards Salmonella. After infection, body weight gain and food intake were higher in the calcium phosphate group. Calcium phosphate and milk Ca decreased faecal enterobacteria before infection. All Ca salts decreased infection-induced intestinal permeability and persistence of Salmonella. Calcium phosphate, milk Ca, calcium carbonate and calcium chloride are able to enhance the intestinal resistance to Salmonella in rats.

Damage to the intestinal epithelial barrier by antibiotic pretreatment of salmonella-infected rats is lessened by dietary calcium or tannic acid.

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.

Dietary calcium decreases but short-chain fructo-oligosaccharides increase colonic permeability in rats.

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.

Supplemental calcium attenuates the colitis-related increase in diarrhea, intestinal permeability, and extracellular matrix breakdown in HLA-B27 transgenic rats.

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.

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats.

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.

Ileal mucosal and fecal pancreatitis associated protein levels reflect severity of salmonella infection in rats.

BACKGROUND: Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection. RESULTS: Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces. CONCLUSIONS: PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marker.

Probiotic Lactobacillus plantarum 299v does not counteract unfavorable phytohemagglutinin-induced changes in the rat intestinal microbiota.

Application of phytohemagglutinin (PHA) in weaning feed has been suggested to stimulate intestinal epithelium maturation. In this study, PHA strongly affected the fecal bacterial population structure of rats. Escherichia coli overgrowth was not prevented by probiotic mannose-adhering Lactobacillus plantarum 299v. Therefore, use of PHA in weaning feed deserves careful evaluation.

Dietary heme adversely affects experimental colitis in rats, despite heat-shock protein induction.

OBJECTIVE: Research on dietary modulation of inflammatory bowel disease is in its infancy. Dietary heme, mimicking red meat, is cytotoxic to colonic epithelium and thus may aggravate colitis. Alternatively, heme-induced colonic stress might also result in potential protective heat-shock proteins (HSPs). Therefore, we investigated the effect of dietary heme on trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. METHODS: Rats were fed a high-fat control diet or a similar diet supplemented with heme. After dietary adaptation, rats were rectally infused with TNBS for colitis induction or saline for sham treatment. Colitis severity was evaluated and several markers were quantified in colonic mucosa isolated 1 wk after colitis induction. Furthermore, cytotoxicity of fecal water and serum α-1-acid glycoprotein were measured. RESULTS: Dietary heme increased cytotoxicity of the fecal water. Heme-fed sham-treated rats had higher colonic HSP-25 and heme-oxygenase-1 mRNA levels, which was confirmed by immunohistochemistry. HSP induction by heme was associated with decreased protein levels of myeloperoxidase and interleukin-1ß after subsequent TNBS infusion. However, no dietary effects were observed on histologic colitis score. Furthermore, body weight gain, colon length, and food intake were lower and α-1-acid glycoprotein concentrations were higher in heme-fed colitic rats. In addition, somatostatin, involved in mucosal repair, was not changed with TNBS infusion in heme-fed rats. CONCLUSION: Dietary heme adversely affects colitis, despite HSP induction. We speculate that the irritating influence of dietary heme, being continuously present in the colon, impairs recovery after colitis induction. A diet high in red meat might be a risk factor for inflammatory bowel disease development.

Supplemental antioxidants do not ameliorate colitis development in HLA-B27 transgenic rats despite extremely low glutathione levels in colonic mucosa.

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.