Toxicological effects of iron on intestinal cells

The aim of the present study was to investigate whether iron, which is involved in the formation of free radicals in vitro, can initiate cellular injury in human intestinal cells. The effects of various concentrations of iron were studied in preconfluent, colonic-cancerogenous cells, and also in postconfluent, differentiating cells. Cellular damage was assessed using cell proliferation (serial cell counting), tetrazolium dye (MTT) uptake, lactate dehydrogenase (LDH) release and apoptosis studies based on caspase-3 activities. Also the activities of the major antioxidative enzymes, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were measured after the cells had been exposed to iron. Our results indicated that preconfluent cells were more susceptible to iron toxicity, as assessed by a significant reduction in cell proliferation and MTT uptake in a concentration-dependent manner compared to the control. However, no evidence for MTT uptake was observed in postconfluent cells. Caspase-3 activity, an indicator of cell apoptosis, considerably increased in preconfluent cells at high iron levels compared to the control (p < 0.05), whereas postconfluent cells were not significantly affected. LDH release was similar for both groups and was significantly higher than the control at 900 microM iron and above. SOD activities were not affected by iron in either group, whereas GPx was considerably higher in iron-treated cells in both groups compared with the control (because of relatively high standard deviations this effect was not significant). In conclusion we suggest that iron exerts its toxic effects intracellularly especially in preconfluent Caco-2 cells, whereas only high iron doses were able to alter the viability of differentiating, enterocyte-like cells.     

The early phase of programmed cell death in Caco-2 intestinal cells exposed to PTH

The regulation of apoptosis is critical for ensuring the homeostasis of an organism. As such, the cell has derived various mechanisms to precisely control the balance between survival and apoptotic signaling. Parathyroid hormone (PTH) function as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. Depending on the cell type involved, PTH also inhibits or promotes the apoptosis. In a previous work we found that PTH promotes the apoptosis of human Caco-2 intestinal cells. In the current study, we demonstrate, for the first time, that stimulation of Caco-2 cells with PTH (10(-8) M) results in the dephosphorylation and translocation of pro-apoptotic protein Bad from the cytosol to mitochondria and release of cytochrome c and Smac/Diablo. The hormone also triggers mitochondria cellular distribution to the perinuclear region, morphological features consistent with apoptosis. PTH increases the enzymatic activity of caspase-3 (48 h) that is also evidenced from the appearance of its cleaved fragments in western blot experiments. Moreover, active caspase-3 is present in nucleus after PTH treatment. In addition, a caspase-3 substrate, poly (ADP-ribose) polymerase (PARP), is degraded by 48 h of PTH treatment. Taken together, our results suggest that, in Caco-2 cells, the induction of apoptosis in response to PTH is mediated by translocation of mitochondria to the perinuclear region, dephosphorylation of Akt, dephosphorylation of Bad and its movement to the mitochondria and subsequent release of cytochrome c and Smac/Diablo which result in activation of downstream caspase-3.     

Blockade of Salmonella enteritidis passage across the basolateral barriers of human intestinal epithelial cells by specific antibody

Antibodies specific to Salmonella enteritidis (S.E.) were obtained from immunized egg yolk, and their protective effects against S.E. were studied by using monolayer-cultured human intestinal epithelial cells, Caco-2 and T84. The Salmonella adherence and entry to the cells were partially inhibited by the antibodies. The antibodies inhibited the decrease in transepithelial electrical resistance (TEER) of the intestinal epithelial monolayers and IL-8 secretion of the cells induced by S.E. invasion. Also, the antibodies blocked the penetration of bacteria through the cell layer although they did not inhibit the growth of bacteria in the cells. Confocal microscopic photographs revealed the bacteria in the infected monolayer cells were bound to antibodies. These results indicate that anti-S.E. antibodies may protect the cells from destruction induced by S.E. invasion in intestinal epithelial cells in addition to the partial inhibition of adhesion and invasion of S.E. at the cell surface. Passive antibodies against invasive bacteria would be useful to prevent the migration of S.E. to blood not only at the cell surface but also inside of intestinal epithelial cells.     

An unusual metabolite of cimicidol-3-O-beta-D-xyloside from Cimicifugae rhizoma by rat intestinal bacteria

Cimicidol-3-O-beta-D-xyloside (1), one of the main components isolated from Cimicifugae rhizoma, is an active component with antiosteoporotic effect. The metabolism of 1 by rat intestinal bacteria was investigated, and two metabolites, 11beta-hydroxycimigenol (2) and foetidinol (3), were isolated and characterized by spectroscopic means including two-dimensional NMR. Furthermore, the structures of six intermediates in the bacterial incubation were proposed on the basis of LC/MS analyses, and the possible metabolic pathway of the formation of 2, which passes through a unique sequence including hydrolysis, hydroxylation, and reduction, is discussed in detail.     

Differential activities of peroxisomes along the mouse intestinal epithelium

The presence of peroxisomes in mammalian intestine has been revealed formerly by catalase staining combined with electron microscopy. Despite the central role of intestine in lipid uptake and the established importance of peroxisomes in different lipid‐related pathways, few data are available on the physiological role of peroxisomes in intestinal metabolism, more specifically, α‐, β‐oxidation, and etherlipid synthesis. Hence, the peroxisomal compartment was analyzed in more detail in mouse intestine. On the basis of immunohistochemistry, the organelles are mainly confined to the epithelial cells. The expression of the classical peroxisome marker catalase was highest in the proximal part of jejunum and decreased along the tract. PEX14 showed a similar profile, but was still substantial expressed in large intestinal epithelium. Immunoblotting of epithelial cells, isolated from the different segments, showed also such gradient for some enzymes, ie, catalase, ACOX1, and D‐specific multifunctional protein 2, and for the ABCD1 transporter, being high in small and low or absent in large intestine. Other peroxisomal enzymes (PHYH, HACL1, and ACAA1), the ABCD2 and ABCD3 transporters, and peroxins PEX13 and PEX14, however, did not follow this pattern, displaying rather constant signals throughout the intestinal epithelium. The small intestine displayed the highest peroxisomal β‐oxidation activity and is particularly active on dicarboxylic acids. Etherlipid synthesis was high in the large intestine, and colonic cells had the highest content of plasmalogens. Overall, these data suggest that peroxisomes exert different functions according to the intestinal segment.     

Dual labeling with 5‐bromo‐2'‐deoxyuridine and 5‐ethynyl‐2'‐deoxyuridine for estimation of cell migration rate in the small intestinal epithelium

Small intestinal epithelium is a self‐renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt‐villus axis. We examined whether dual labeling with different thymidine analogues, 5‐bromo‐2'‐deoxyuridine (BrdU) and 5‐ethynyl‐2'‐deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the “click” reaction, respectively. We successfully observed BrdU‐ and EdU‐positive cells on the epithelium with no cross‐reaction. In addition, we observed an almost complete overlapping of BrdU‐ and EdU‐positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU‐ and EdU‐positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5 μm/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.     

Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2

The human intestinal cell line Caco-2 is a well-established model system to study cellular differentiation of human enterocytes of intestinal origin, because these cells have the capability to differentiate spontaneously into polarized cells with morphological and biochemical features of small intestinal enterocytes. Therefore, the cells are widely used as an in vitro model for the human intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of intestinal cellular differentiation. The proteome of proliferating Caco-2 cells was compared with that of fully differentiated cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon cellular differentiation. In parallel, proteins associated with proliferation, cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis--diseases associated with an imbalanced differentiation process of intestinal cells.     

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 μM), and, less potently, by Caco-2 cells (IC(50) = 402 μM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 μM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.     

Differentiation potential of intestinal mesenchyme and its interaction with epithelial cells: a study using beta-galactosidase-expressing fibroblast lines

Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either beta-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene was more efficient than electroporation. The expression of beta-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of beta-galactosidase were isolated. These clones expressed beta-galactosidase even after prolonged passage in the absence of selection. The beta-galactosidase tagged lines (F1:G9gal and A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9gal and A1:F1gal cells differed in their morphological organisation but this did not change their expression of smooth muscle alpha-actin.     

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.     

Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors

BACKGROUND AND AIMS: While mature enterocytes are resistant to transduction by adenovirus type 5 (Ad5) vectors, undifferentiated cells are transduced much more efficiently. Our purpose was to study enterocyte transduction in models of intestinal wound healing. METHODS: Transduction was studied ex vivo using cultures of endoscopic biopsies and in vitro utilizing Caco-2 cells in models of mucosal wound healing. Vectors carried either the LacZ or the luciferase gene. CAR (coxsackievirus and adenovirus receptor) and integrins were studied with transduction inhibition and immunofluorescent staining. RESULTS: Increased transduction efficiency was observed for a subset of enterocytes with a flattened de-differentiated phenotype present at the edge of cultured biopsies. In the in vitro systems, expanding Caco-2 cell monolayers exhibited increased transducibility that was time- and dose-dependent, reaching virtually 100% in cells along the leading edge at high viral load. Bioluminescence activity of transduced expanding monolayers was up to 3-fold greater than that of non-expanding monolayers ('fence' system, 48 h, MOI 1000, p < 0.05). Mitomycin C pre-treatment did not affect levels of transduction in expanding monolayers. At the highest viral load tested, CAR or integrin blocking prior to virus application resulted in 39.4% and 45.4% reduction in transduction levels (p < 0.05). Immunofluorescence revealed altered expression of CAR on the migrating edge of the Caco-2 cultures and the expression of CAR on the apical membrane of biopsy enterocytes. CONCLUSIONS: Increased CAR and integrin accessibility in migrating enterocytes mediates increased transduction by Ad5 vectors. This subset of enterocytes provides a target for the delivery of genes of interest for both research and gene therapy applications.     

Outer membrane protein OmpV mediates Salmonella enterica serovar typhimurium adhesion to intestinal epithelial cells via fibronectin and α1β1 integrin

Salmonella typhimurium is an invasive Gram‐negative enteric bacterium, which causes salmonellosis, a type of gastroenteritis in humans and typhoid‐like symptoms in mice. Upon entering through the contaminated food and water, S. typhimurium adheres, colonises, and invades intestinal epithelial cells (IECs) of the small intestine. In this study, we have shown that upon deletion of the outer membrane protein OmpV, there is a significant decrease in adherence of S. typhimurium to the IECs, indicating that OmpV is an important adhesin of S. typhimurium. Further, our study showed that OmpV binds to the extracellular matrix component fibronectin and signals through α1β1 integrin receptor on the IECs and OmpV‐mediated activation of α1β1, resulting in the activation of focal adhesion kinase and F‐actin modulation. Actin modulation is crucial for bacterial invasion. To the best of our knowledge, this is the first report of an adhesin mediated its effect through integrin in S. typhimurium. Further, we have observed a decrease in pathogenicity in terms of increased LD₅₀ dose, lesser bacterial numbers in stool, and less colonisation of bacteria in different organs of mice infected with Δompv mutant compared with the wild‐type bacteria, thus confirming the crucial role of OmpV in the pathogenesis of S. typhimurium.     

Irisin reverses intestinal epithelial barrier dysfunction during intestinal injury via binding to the integrin αVβ5 receptor

Disruption of the gut barrier results in severe clinical outcomes with no specific treatment. Metabolic disorders and destruction of enterocytes play key roles in gut barrier dysfunction. Irisin is a newly identified exercise hormone that regulates energy metabolism. However, the effect of irisin on gut barrier function remains unknown. The therapeutic effect of irisin on gut barrier dysfunction was evaluated in gut ischemia reperfusion (IR). The direct effect of irisin on gut barrier function was studied in Caco‐2 cells. Here, we discovered that serum and gut irisin levels were decreased during gut IR and that treatment with exogenous irisin restored gut barrier function after gut IR in mice. Meanwhile, irisin decreased oxidative stress, calcium influx and endoplasmic reticulum (ER) stress after gut IR. Moreover, irisin protected mitochondrial function and reduced enterocyte apoptosis. The neutralizing antibody against irisin significantly aggravated gut injury, oxidative stress and enterocyte apoptosis after gut IR. Further studies revealed that irisin activated the AMPK‐UCP 2 pathway via binding to the integrin αVβ5 receptor. Inhibition of integrin αVβ5, AMPK or UCP 2 abolished the protective role of irisin in gut barrier function. In conclusion, exogenous irisin restores gut barrier function after gut IR via the integrin αVβ5‐AMPK‐UCP 2 pathway.     

A tissue‐engineered model of the intestinal lacteal for evaluating lipid transport by lymphatics

Lacteals are the entry point of all dietary lipids into the circulation, yet little is known about the active regulation of lipid uptake by these lymphatic vessels, and there lacks in vitro models to study the lacteal—enterocyte interface. We describe an in vitro model of the human intestinal microenvironment containing differentiated Caco‐2 cells and lymphatic endothelial cells (LECs). We characterize the model for fatty acid, lipoprotein, albumin, and dextran transport, and compare to qualitative uptake of fatty acids into lacteals in vivo. We demonstrate relevant morphological features of both cell types and strongly polarized transport of fatty acid in the intestinal‐to‐lymphatic direction. We found much higher transport rates of lipid than of dextran or albumin across the lymphatic endothelial monolayer, suggesting most lipid transport is active and intracellular. This was confirmed with confocal imaging of Bodipy, a fluorescent fatty acid, along with transmission electron microscopy. Since our model recapitulates crucial aspects of the in vivo lymphatic–enterocyte interface, it is useful for studying the biology of lipid transport by lymphatics and as a tool for screening drugs and nanoparticles that target intestinal lymphatics. Biotechnol. Bioeng. 2009;103: 1224–1235. © 2009 Wiley Periodicals, Inc.     

Isolation of catechin-converting human intestinal bacteria

Aims: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. Methods and Results: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin‐converting human faecal suspension. The isolates catalysed individual steps in the degradation of (?)‐epicatechin and (+)‐catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C‐ring of both (?)‐epicatechin and (+)‐catechin giving rise to 1‐(3,4‐dihydroxyphenyl)‐3‐(2,4,6‐trihydroxyphenyl)propan‐2‐ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1‐(3,4‐dihydroxyphenyl)‐3‐(2,4,6‐trihydroxyphenyl)propan‐2‐ol to 5‐(3,4‐dihydroxyphenyl)‐γ‐valerolactone and 4‐hydroxy‐5‐(3,4‐dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. Conclusions: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. Significance and Impact of the Study: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin‐converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.