The Caco-2 cell monolayer model is widely used as a standard screening tool for studying the mechanisms of cellular drug transport. Caffeine was chosen as a model drug and is supposed to be class I of the Biopharmaceutics Classification System (BCS). Our study was conducted 1) to characterize the mechanisms of caffeine transport across the intestinal barrier, 2) to classify caffeine according to BCS, 3) to predict drugs intestinal absorption in humans. METHODS: Caffeine transport (0.1, 0.3, 1 and 10 mmol/l) was studied in Caco-2 cell monolayer in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under iso-pH 7.4 and pH-gradient (6/7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+- free transport medium (opening tight junctions). RESULTS: The caffeine transport was linear with time, transport direction and pH independent, displaying non-saturable (first-order) kinetics, with high permeability coefficient (Papp): in AP-BL direction Papp = 46.3-53.5 x 10-6 cm/s; in BL-AP direction Papp = 45.6-49.4 x 10-6 cm/s. Thus, the transport seems to be transcellular mediated by passive diffusion. Using Ca2+- free transport medium tight junctions were opened (confirmed by increased Papp of mannitol) but the caffeine Papp was not changed. Thus, the paracellular route is only a minor way of caffeine transport. CONCLUSION: High solubility and high permeability of caffeine rank it among class I of BCS and well absorbed compounds

• MeSH
• biologický transport fyziologie   účinky léků   MeSH
• buněčné kultury MeSH
• Caco-2 buňky MeSH
• difuze MeSH
• financování organizované MeSH
• intestinální absorpce MeSH
• kinetika MeSH
• kofein farmakokinetika   farmakologie   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• lineární modely MeSH
• mannitol metabolismus   MeSH
• permeabilita účinky léků   MeSH
• střevní sliznice metabolismus   MeSH
• těsný spoj metabolismus   účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• xenobiotika farmakokinetika   farmakologie   MeSH
• Check Tag
• lidé MeSH

Experimental data concerning the bioavailability of the different Mg-salts in human organism is inconsistent. Mg-absorption reported by clinical studies largely varies depending on the method used for evaluation. The aim of this study was to evaluate the bioavailability and accessibility of magnesium bound in different Mg-salt compounds, using an in vitro model of intestinal cell barrier. The study included a variety of inorganic (oxide, sulphate, chloride, carbonate) and organic salts (lactate, citrate, pidolate). Caco-2 cells were cultivated in a complete culture medium with different magnesium salts treatments in ascending concentrations. The viability and quantity of cells was analysed by FACS. Mg-absorption was analysed by a direct colorimetric assay, measured by spectrometry. T-test identified a significant decrease in cell count treatment with mg-lactate compared with citrate. Mg-pidolate showed a significantly higher cell viability compared with Mg-citrate, Mg-lactate and Mg-chloride. Even though the difference was not significant, we showed that an increase in Mg2+ salt concentration progressively decreased the cell count and the viability and the effect was universal for all the used Mg-salt treatments. Mg-citrate, chloride, and sulphate showed a significantly lower absorption compared to Mg-carbonate, pidolate and oxide. Our in vitro monolayer model of human intestinal transport showed that viability and quantity of cell decreased with increasing Mg-concentration. We admit that our experiment model may have some limitations in accurately describing an in vivo Mg2+ absorption. Moreover, it is also necessary to assess the relevance of our data in vivo and especially in clinical practice.

• MeSH
• Caco-2 buňky MeSH
• hořčík metabolismus   MeSH
• lidé MeSH
• střevní sliznice metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The determination of mycotoxins content in food is not sufficient for the prediction of their potential in vivo cytotoxicity because it does not reflect their bioavailability and mutual interactions within complex matrices, which may significantly alter the toxic effects. Moreover, many mycotoxins undergo biotransformation and metabolization during the intestinal absorption process. Biotransformation is predominantly the conversion of mycotoxins meditated by cytochrome P450 and other enzymes. This should transform the toxins to nontoxic metabolites but it may possibly result in unexpectedly high toxicity. Therefore, the verification of biotransformation and bioavailability provides valuable information to correctly interpret occurrence data and biomonitoring results. Among all of the methods available, the in vitro models using monolayer formed by epithelial cells from the human colon (Caco-2 cell) have been extensively used for evaluating the permeability, bioavailability, intestinal transport, and metabolism of toxic and biologically active compounds. Here, the strengths and limitations of both in vivo and in vitro techniques used to determine bioavailability are reviewed, along with current detailed data about biotransformation of mycotoxins. Furthermore, the molecular mechanism of mycotoxin effects is also discussed regarding the disorder of intestinal barrier integrity induced by mycotoxins.

• MeSH
• biologická dostupnost MeSH
• Caco-2 buňky MeSH
• epitelové buňky enzymologie   MeSH
• hodnocení rizik MeSH
• intestinální absorpce * MeSH
• lidé MeSH
• metabolická aktivace MeSH
• metabolická inaktivace MeSH
• mykotoxiny metabolismus   toxicita   MeSH
• permeabilita MeSH
• střevní sliznice enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Pseudobutyrivibrio xylanivorans strain Mz5(T), an anaerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach, i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability of P. xylanivorans Mz5(T). The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.

• MeSH
• anaerobní bakterie izolace a purifikace   růst a vývoj   MeSH
• bachor mikrobiologie   MeSH
• bakteriální adheze fyziologie   genetika   imunologie   MeSH
• Caco-2 buňky enzymologie   mikrobiologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• kyselina máselná izolace a purifikace   metabolismus   MeSH
• kyselina mléčná izolace a purifikace   metabolismus   MeSH
• Lactobacillus metabolismus   růst a vývoj   MeSH
• probiotika izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

Puwainaphycins (PUW) and minutissamides (MIN) are cyanobacterial lipopeptides found in various cyanobacterial species. The first possible target of human exposure to them is intestinal epithelium but effect of PUW/MIN on enterocytes is not known at all. Using differentiated Caco-2 cells, PUW F was found to be cytotoxic from 5 µM concentration based on lactate dehydrogenase release assay and total protein concentration. However, it is also able to induce production of interleukin 8 in non-cytotoxic concentrations 1 and 2.5 µM detected by ELISA. Effects of MIN A and C were similar but less pronounced compared to PUW F. On the other hand, MIN D was the least toxic compound with no significant pro-inflammatory effects. Surprisingly, pro-inflammatory activation of the cells by PUW F and MIN C resulted in an increase in tight junction (TJ) protein claudin 4 expression determined by western blot analysis and confirmed by confocal microscopy. Furthermore, decrease in expression of zonula occludens 3, another TJ protein, was observed after the exposure to PUW F. Taken together, these cytotoxic lipopeptides, especially PUW F, are to be studied more deeply due to their capability to activate and/or deregulate human enterocytes in low concentrations.

• MeSH
• Caco-2 buňky MeSH
• lidé MeSH
• lipopeptidy * MeSH
• sinice * MeSH
• střevní sliznice MeSH
• těsný spoj MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

• MeSH
• biokompatibilní materiály chemie   farmakologie   MeSH
• buňky Hep G2 MeSH
• Caco-2 buňky MeSH
• hydrofobní a hydrofilní interakce * MeSH
• lidé MeSH
• lipidy chemie   farmakologie   MeSH
• nanočástice chemie   MeSH
• povrchové vlastnosti MeSH
• sérový albumin chemie   MeSH
• termodynamika MeSH
• velikost částic MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE) and epigallocatechin gallate (EGCG) using both proliferating (P) and differentiated (D) caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes' activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST) activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT) activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well.

• MeSH
• buněčná diferenciace účinky léků   MeSH
• Caco-2 buňky MeSH
• čaj chemie   MeSH
• glutathiontransferasa biosyntéza   MeSH
• katechin analogy a deriváty   chemie   farmakologie   MeSH
• katechol-O-methyltransferasa biosyntéza   MeSH
• lidé MeSH
• messenger RNA biosyntéza   MeSH
• proliferace buněk účinky léků   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Flavonoids, including anthocyanins, are polyphenolic compounds present in fruits, vegetables and dietary supplements. They can be absorbed from the intestine to the bloodstream or pass into the large intestine. Various bacterial species and enzymes are present along the entire intestine. The aim of the present work was to investigate the intestinal metabolism of selected dietary polyphenol and polyphenol glycosides (quercetin, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and delphinidin-3-O-galactoside) by human fecal bacteria. Moreover, the metabolism of metabolites formed from these compounds in human colon carcinoma cells (Caco-2) was also point of the interest. Test compounds were added to fresh human stool in broth or to Caco-2 cells in medium and then incubated for 6 or 20 h at 37°C. After incubation, samples were prepared for LC/MS determination. Main metabolic pathways were deglycosylation, hydrogenation, methylation, hydroxylation, and decomposition. 2,4,5-trihydroxybenzaldehyde, as a metabolite of cyanidin glycosides, was detected after incubation for the first time. Metabolites formed by fecal bacteria were further glucuronidated or methylated by intestinal enzymes. This metabolite profiling of natural compounds has helped to better understand the complex metabolism in the human intestine and this work also has shown the connection of metabolism of natural substances by intestinal bacteria followed by metabolism in intestinal cells.

• MeSH
• Bacteria metabolismus   MeSH
• Caco-2 buňky MeSH
• feces mikrobiologie   MeSH
• flavonoidy metabolismus   MeSH
• glykosidy metabolismus   MeSH
• lidé MeSH
• metabolické sítě a dráhy MeSH
• metabolom * MeSH
• nádory tračníku metabolismus   MeSH
• polyfenoly metabolismus   MeSH
• střevní sliznice metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10(6) to 10(9) CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.

• MeSH
• bakteriální adheze MeSH
• Caco-2 buňky MeSH
• Escherichia coli fyziologie   MeSH
• feces mikrobiologie   MeSH
• Lactobacillus fyziologie   izolace a purifikace   MeSH
• lidé MeSH
• novorozenec MeSH
• probiotika izolace a purifikace   MeSH
• Salmonella typhi fyziologie   MeSH
• střeva mikrobiologie   MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH

• MeSH
• biologické modely MeSH
• biologický transport MeSH
• Caco-2 buňky fyziologie   MeSH
• financování organizované MeSH
• intestinální absorpce MeSH
• permeabilita MeSH
• preklinické hodnocení léčiv metody   MeSH