Enhancement of D-chiro-inositol transport across intestinal cells by alpha-Lactalbumin peptides.

OBJECTIVE: This study aims to characterize in vitro D-chiro-inositol intestinal absorption and identify factors able to improve its bioavailability. D-chiro-inositol, one of the natural occurring stereoisomer of myo-inositol, acts as a second messenger in insulin-regulated glucose metabolism in complementary mode with myo-inositol. Because of their insulin-mimetic activities and safety, both myo-inositol and D-chiro-inositol are often employed as supplements in insulin-resistance treatment. MATERIALS AND METHODS: Trans-epithelial passage of D-chiro-inositol was evaluated in the human intestinal Caco-2 cell line differentiated on filter, a widely established in vitro model to study intestinal absorption. D-chiro-inositol transport was assayed in a concentration range corresponding to an estimated in vivo concentration following oral supplementation. α-Lactalbumin peptides, obtained by in vitro simulated gastrointestinal digestion, were tested as possible modulators of the intestinal permeability of D-chiro-inositol. RESULTS: The absorption of this stereoisomer was relatively low and presumably due to passive diffusion, while it was greatly enhanced by the presence of α-Lactalbumin digest. α-Lactalbumin peptides induced an increase in paracellular permeability that was completely reversible, indicating lack of cytotoxicity. This effect involved temporary rearrangement of F-actin apical cytoskeleton and of the tight junction protein ZO-1. CONCLUSIONS: Although further studies are required to identify and characterize the most effective peptides, the ability of α-Lactalbumin digest to act as absorption enhancers may have very interesting and promising applications in the fields of nutritional supplements and pharmacology.

Intracellular zinc stores protect the intestinal epithelium from Ochratoxin A toxicity.

Ochratoxin A (OTA) is a harmful mycotoxin frequently contaminating foods, feeds and beverages. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet poorly characterized mechanisms. Although intestinal cells are relatively resistant to high concentrations of OTA, interaction with other dietary factors or specific nutritional conditions may increase OTA toxicity to the intestinal mucosa. The role of intracellular zinc stores in protecting the integrity of intestinal mucosa has been investigated in human Caco-2/TC7 cells challenged with OTA. Zinc depletion of cells incubated with TPEN, a specific zinc chelator, caused an increase of tight junction permeability in OTA treated cells, accompanied by increased apoptosis. These effects were fully reverted by zinc supplementation during TPEN treatment, showing a specific role for this micronutrient in enterocyte defence mechanisms from OTA toxicity. A complex perturbation of zinc homeostasis was also demonstrated by analyzing the expression of genes coding for proteins involved in cellular zinc. In particular, zinc-dependent up-regulation of the metallothionein gene MT2A upon OTA treatment may indicate that the mycotoxin acts through generation of redox imbalance and that zinc deprivation reduces the intracellular defence mechanisms against noxious insults.

Effects of red wine on ochratoxin A toxicity in intestinal Caco-2/TC7 cells.

Ochratoxin A (OTA) is found in a variety of foods and beverages, including red wine. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet uncharacterized mechanisms. Consumption of contaminated wines might contribute up to 13% of OTA daily human intake. Potentially chronic exposure has therefore raised public health concern. OTA toxicity in the presence of de-alcoholated red wine was investigated in human intestinal Caco-2/TC7 cells, differentiated on filter supports, by measuring tight junction (TJ) permeability, morphological alterations of TJ proteins and occurrence of apoptosis. Cells were treated with OTA, in the presence of de-alcoholated red wine, for 48h and the ability to recover from the effects of OTA was evaluated after 24h in complete medium. OTA treatment increased TJ permeability and caused intracellular redistribution of claudin-4. However, cells were able to restore permeability and correct localization of claudin-4 following 24h recovery. Conversely, in the presence of red wine, OTA produced faster and irreversible increase in TJ permeability, intracellular delocalization of claudin-4 and extensive apoptosis. Our results point at a possible synergy between OTA and some red wine components, such as polyphenols, in the induction of apoptotic cell death.

The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

Biphasic effect of iron on human intestinal Caco-2 cells: early effect on tight junction permeability with delayed onset of oxidative cytotoxic damage.

Treatment of differentiated human intestinal Caco-2 cells with Fe(II) ascorbate altered tight junction permeability in a dose and time-dependent way for up to 3 hr of treatment Upon iron removal and transfer to complete culture medium, the effect was reversible up to 10 microM Fe(II), while at higher concentrations a late phase toxic effect was observed. Reduction of intracellular energy abolished the short term effect of iron on tight junction permeability without affecting its cellular uptake, suggesting that active processes, other than transport, were involved. The short term effect of iron the permeability of tight junctions did not appear to result from the generation of reactive oxygen species, as it was not prevented by antioxidant treatment under normal energy conditions. Conversely, the late phase effect leading to both apoptosis and necrosis during the 24 hr following iron removal could be reduced by antioxidant treatment and was exacebated by GSH depletion. Iron induced oxidative stress may therefore be responsible for membrane damage and cellular death occurring in the late phase. The reported effects of iron on intestinal tight junction permeability followed by more widespread cytotoxicity from oxidative events should be considered in light of the extensive use of iron supplementation in different phases of human life.

Iron and copper alter tight junction permeability in human intestinal Caco-2 cells by distinct mechanisms.

Human intestinal Caco-2 cells differentiated for 15-17 days on transparent filter inserts were treated for up to 3 h with 50 and 100 microM CuCl(2) or FeSO(4) in the AP compartment at pH 6.0. Trans-epithelial electrical resistance (TEER) showed a progressive decrease during the course of the experiment that was slower in cells treated with 50 microM CuCl(2) than in those treated with 100 microM CuCl(2). Both 50 and 100 microM FeSO(4) produced a similar decrease in TEER over time, tailing off after 120 min. F-actin localization by fluorescent phalloidin binding in control cells and in cells treated for 3 h with 50 microM CuCl(2) or FeSO(4) highlighted striking differences in the two treatments. Cu(II) led to an overall reduction in F-actin staining with extensive depolymerization in areas of the monolayer, in the absence of cellular loss. Conversely, Fe(II) treatment produced disorganization of F-actin and decreased staining of the perijunctional actin filaments. No changes in the localization and intensity of staining of the junctional proteins ZO1, occludin and E-cadherin were observed after treatment with 100 microM FeSO(4) in analogy with previous observations in Cu(II)-treated cells. The data presented suggest that different mechanisms are responsible for the changes to tight junction permeability produced by the two metals.

Copper uptake and intracellular distribution in the human intestinal Caco-2 cell line.

The apical uptake of 64CuCl2 was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH 6.0 in the apical compartment, the uptake of copper was linear over the first 6 min and between 10 and 80 microM CuCl2 exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10 mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake ('pulse') and of 'chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione). and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.

Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells.

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10-100 microM CuCl(2) (up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to L-histidine [Cu(His)(2)]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of (64)CuCl(2), both exhibiting a maximum at pH 6.0. Treatment with CuCl(2) produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 microM Cu(His)(2) for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.

Carbonic anhydrase I reduction in colonic mucosa of patients with active ulcerative colitis.

Ulcerative colitis (UC) is associated with low intracolonic pH and unbalanced transmucosal ionic exchanges. Along the gastrointestinal tract carbonic anhydrase isoenzyme I (CA-I) is specifically expressed in colon epithelium and is involved in mucosal control of ion, fluid, and acid-base balance. Since altered CA-I expression may play some role in UC, CA-I was measured at the mRNA and protein level and carbonic anhydrase (CA) enzyme activity was determined in colon biopsies of 14 UC patients (6 remission, 4 mild, 4 moderate UC) and of 12 healthy subjects. Patients with mild or moderate UC showed a significant reduction of CA-I mRNA and protein and of total CA activity in the inflamed mucosa compared to controls. Patients with UC in remission showed a pattern of CA-I expression and CA activity similar to controls. This is the first report showing a reduction in the expression of CA-I in active UC.

Effects of retinoids on gene expression in different epithelial models in vivo and in vitro.

We have previously reported that the induction of Vitamin A deficiency results in a threefold decrease in the hepatic expression of cellular retinol binding protein I (CRBP I) mRNA in vivo and that the treatment of intestinal cell lines in vitro with retinoids leads to the induction of CRBP I transcription. In the present paper we extend the analysis to retinoid-dependent gene expression in the testicular epithelium in vivo and in the intestinal cell line FRIC B. In rat testis excess Vitamin A results in the reduced production of mature spermatozoa and in the premature release of immature germ cells in the lumen, while Vitamin A deficiency leads to almost complete degeneration of the germinal epithelium. We show reduced level of expression of CRBP I mRNA in vitamin A deficient testis. Retinoid treatment of cultured intestinal cells, which induces a reorganization of the actin cytoskeleton, has no effect on the expression of the differentiation induced gene Dri 42. The results show that even though unable to trigger by themselves the differentiation process, retinoids exert a direct effect on the expression of specific genes.

The efflux of lysine from the basolateral membrane of human cultured intestinal cells (Caco-2) occurs by different mechanisms depending on the extracellular availability of amino acids.

The efflux of the nutritionally essential amino acid, L-lysine from the basolateral (BL) membrane was characterized in human cultured intestinal cells (Caco-2) grown and differentiated on permeable filter supports. Cells were loaded by incubating with 3H-lysine from the apical (AP) side in the absence of sodium (substituted with choline) in the BL medium; under these conditions, cells accumulated lysine in the intracellular soluble pool to 10- to 20-fold the extracellular concentration. L-Lysine efflux in the BL medium was then followed, and initial rates of efflux were calculated under different experimental conditions. L-Lysine efflux exhibited a strong energy dependence. The presence of an inwardly directed gradient of sodium or lithium stimulated lysine efflux; ouabain reduced efflux in both sodium- and lithium-containing medium. When zwitterionic or cationic amino acids were added to the BL medium, L-lysine efflux was strongly stimulated. The most efficient trans-stimulating amino acids were L-leucine > L-methionine = L-ornithine = L-arginine. In the presence of trans-stimulating amino acids in the BL medium, L-lysine efflux exhibited energy independence and was not affected by the presence of a sodium gradient. In addition, the sensitivity, of efflux to N-ethylmaleimide was different in the absence or in the presence of amino acids in the BL medium. These results suggest that different mechanisms may operate in the BL efflux of L-lysine from human intestinal epithelial cells, depending on the extracellular availability of other amino acids, to guarantee optimal bioavailability of this essential amino acid both in the postprandial absorptive period and between meals.

Transport of the antibacterial agent oxazolidin-2-one and derivatives across intestinal (Caco-2) and renal (MDCK) epithelial cell lines.

The transepithelial passage of the orally bioavailable antibacterial agent oxazolidin-2-one (OXa) and 10 derivatives has been studied with human intestinal (Caco-2) and canine renal (MDCK) cell lines grown on polycarbonate filters. The transepithelial passage was assayed in the apical-to-basolateral (AP-to-BL) direction and in the opposite direction (BL to AP) in both cell lines. The observed passage rates of OXa were similar in both directions in the two cell lines, suggesting passive diffusion. This was further confirmed by the fact that transport kinetics were linear as a function of initial concentration. The rates of AP-to-BL passage of OXa and seven of the derivatives in both cell lines were linearly related to lipophilicity, whether expressed as high-passage liquid chromatography retention time or as the logarithm of the n-octanol-water partition coefficient (log P). These data suggest that the lipophilicity of OXa is important for its observed bioavailability after oral administration. Interestingly, three of the derivatives exhibited a higher passage rate than predicted by lipophilicity. Further studies indicated that this transport was saturable, similar in the two directions, and not affected by energy depletion, suggesting the presence of an additional carrier-mediated facilitated-transport mechanism.

Characterization of the mucins produced by normal human colonocytes in primary culture.

Mature goblet cells filled with mucin ready for secretion represent about one third of the cells in primary cultures of human colonocytes. In the present study characterization of the mucins produced by cultured human colonocytes was made by histochemical methods by lectin and monoclonal antibody binding. Two monoclonal antibodies and three lectins (Dolichos biflorus (DBA), Helix pomatia (HPA) and Arachis hypogea (PNA) recognizing epitopes or sugar haptens characteristic of different stages of mucin glycoprotein maturation, were employed. The reactivity to these probes was tested both on cultured colonocytes and on tissue sections of the normal colon mucosa. The results show that the mucins produced in culture are glycosylated to the mature form, as they show the same reactivity to lectins and antibodies of the mucins expressed in tissue sections of the normal colon mucosa. In addition, it is demonstrated that cultured human colonocytes do not express mucins reactive to PNA, which are characteristic of tumors. Since the cultured colonocytes maintain the expression of differentiated functions for at least three days, they may offer a useful model to study metabolism, function and regulation of colon mucins in health and disease.

The transport of lysine across monolayers of human cultured intestinal cells (Caco-2) depends on Na(+)-dependent and Na(+)-independent mechanisms on different plasma membrane domains.

To characterize the mechanisms involved in the intestinal absorption of the essential amino acid L-lysine from the diet, the transepithelial transport of L-lysine was studied in monolayers of cultured human intestinal cells (Caco-2) grown and differentiated on microporous membrane supports. L-lysine was transported mainly in the apical (AP) to basolateral (BL) direction and the BL to AP transport was approximately one order of magnitude lower at all concentrations tested. Non-linear regression analysis of the transport in the AP to BL and the BL to AP direction identified, in both cases, single saturable components with similar Km but different Vmax and a nonsaturable diffusional component. The AP to BL L-lysine transport was highly energy- and sodium-dependent and was unaffected by an unfavorable concentration gradient. Selective replacement of sodium ions in the AP or the BL compartment and determination of both AP to BL transport and the intracellular soluble lysine pool showed that uptake occurs via a sodium-independent mechanism, not significantly influenced by membrane potential, whereas efflux is a sodium-dependent process. Competition experiments showed that L-lysine uptake is highly stereospecific and is shared by cationic and large neutral amino acids. This study demonstrates the presence of a sodium-dependent mechanism of lysine efflux across the BL membrane of intestinal cells, which may be essential for lysine transport into the blood circulation. Overall, these results support the use of the Caco-2 cell model for studies of intestinal nutrient transport.

D-cycloserine uses an active transport mechanism in the human intestinal cell line Caco 2.

In a previous study we have shown that cultured epithelial cell lines can be used to measure the transepithelial passage of antimicrobial agents across the intestine and to obtain information on the mechanisms of transport utilized and predict the bioavailability of the antimicrobial agents after oral administration. In particular, among the drugs investigated, D-cycloserine had been shown to be transported in a polarized manner only in the intestinal cells. In the present work, further characterization of the transport of D-cycloserine in the human intestinal cell line Caco 2 has shown that this occurs in the apical-to-basolateral direction by an active mechanism which is energy dependent but only partially sodium dependent. Competition studies have also indicated that the transport of D-cycloserine occurs via a carrier for imino acids, amino acids with aliphatic side chains (L-Ala, D-Ala, and beta Ala), and L-Trp, L-Tyr, L-Cys, and alpha-amino isobutyric acid. This system may correspond to a proton-dependent system for L-proline and beta-alanine recently described for Caco 2 cells. In contrast with the cephalosporins, which are taken up by the Caco 2 cells via a dipeptide carrier, D-cycloserine transport cannot be inhibited by either cephalexin (a member of the class of cephalosporins) or dipeptides.

Human colonocytes in primary culture: a model to study epithelial growth, metabolism and differentiation.

The purpose of this work was to set up an in vitro model for the study of normal and pathological functions of the colonic epithelium. We have isolated colonic crypts by mild proteolytic digestion and mechanical dissociation of human biopsy material obtained during colonoscopy. The crypts, free of connective tissue, when placed in culture rapidly attached to the substrate and formed colonies containing over 95% of epithelial cells. Histochemical and ultrastructural characterization of the colonies showed the presence of both absorptive and secretory cells, exhibiting a high degree of differentiation. Proliferative activity occurred mostly during the first 24 h and progressively declined thereafter. The cells survived and maintained differentiated characteristics for at least three days in culture. This method can be used to study normal functions of the colonic epithelium. It may also be employed to investigate both noxious and protective factors in pathological conditions such as inflammatory bowel disease and colorectal neoplasia.

Expression of epithelial markers and retinoid-binding proteins in retinol- or retinoic acid-treated intestinal cells in vitro.

This study was undertaken with the aim of investigating the effects of retinoids on the expression of differentiated traits in intestinal cell models. The cell lines used included epithelial cells isolated from fetal rat intestines (FRIC), displaying a relatively undifferentiated phenotype, and the human colon adenocarcinoma cell lines Caco 2 and HT29, which express some of the traits of the mature enterocytes under defined culture conditions. The effects of retinoids were also studied in organ cultures of fetal rat intestine, where the epithelial-mesenchymal interactions are preserved. All-trans-retinol and all-trans-retinoic acid treatments were compared in their ability to regulate the expression of genes coding for proteins involved in retinoid metabolism and for cytoskeletal proteins. The results have shown that the effects of the two retinoids were qualitatively similar. A specific induction of the cellular retinol-binding protein CRBP I mRNA was observed following retinoid treatment in one of the two FRIC lines examined (FRIC B) and in organ culture. The expression of the retinoic acid receptors RAR alpha and gamma was not affected by treatment in any of the cultures examined, while RAR beta was expressed only by the organ cultures and was transcriptionally induced by retinoic acid treatment. The retinoids also induced a reorganization of the actin cytoskeleton in the FRIC B cell line, accompanied by a decrease in the expression of two components of the microvillar cytoskeleton, ezrin and villin. The results obtained in both cell and organ cultures suggest that retinoids alone are not able to trigger the differentiation program in the intestinal epithelial cell, irrespective of the level of differentiation already achieved at the time of treatment.

Cytotoxic activity of human lymphocytes against differentiated intestinal tumour cell lines.

The natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activity of human peripheral blood lymphocytes (PBL) against various human tumour cell lines from intestinal origin (WIDR, HT29, Caco-2) has been investigated. The differentiated Caco-2 cells were then used as a model to investigate the cytotoxic activity against enterocyte-like target cells. Caco-2 were seeded on polycarbonate filters and maintained in culture for at least 15 days to allow the differentiation and formation of tight junctions. The integrity of tight junctions was assayed by measuring [3H]mannitol flux from apical to basolateral compartment. Cytotoxic analysis showed that both differentiated and undifferentiated Caco-2 cells were similarly susceptible to NK and LAK activity. The capacity of cytotoxic lymphocytes to kill enterocyte-like cells with intact junctional complex may suggest a direct role of cytotoxic lymphocytes in causing intestinal lesions under inflammatory conditions.

Epithelial cells in culture as a model for the intestinal transport of antimicrobial agents.

The bioavailabilities of orally administered drugs depend to a great extent on their capability of being transported across the intestinal mucosa. In an attempt to develop an in vitro model for studying the intestinal transport of drugs, we used an intestinal epithelial cell line (Caco 2) derived from a human colon adenocarcinoma. A renal epithelial cell line (MDCK) was also used to determine the tissue specificity of drug transport. These cell lines, which were grown on filters, form a monolayer of well-polarized cells coupled by tight junctions and can be used for transcellular transport experiments. We studied the transport of nine antimicrobial agents with different physicochemical and pharmacokinetic characteristics using these epithelial cell monolayers to determine whether this model could be predictive of oral bioavailability. The transepithelial passage was assayed from the apical (AP) to the basolateral (BL) side and in the opposite direction (BL to AP) in both cell lines. Radioactively labeled mannitol was used to monitor the intactness of the cell monolayer during drug passage. The results indicated that all antimicrobial agents tested tended to behave in vitro generally according to their known in vivo absorptive characteristics. In addition, the use of epithelia from different tissues enabled us to divide the drugs into four groups according to their behaviors and suggested the existence of different transport mechanisms. In particular, two antibiotics, gentamicin and teicoplanin, showed no passage in either direction or cell line, in accordance with their very poor in vivo absorbances after oral administration. In contrast, rifapentine, rifampin, and nalidixic acid passed very efficiently at similar rates in both directions and cell lines in a concentration-dependent, nonsaturable manner, which is suggestive of passive diffusion down a concentration gradient. Of the remaining drugs, isoniazid and novobiocin sodium showed some differences in passage between the two cell lines and, given their ionized state at the pH that was used, may use the paracellular route. Finally, trimethoprim and D-cycloserine exhibited differences in passage both with respect to polarity and cell line; in particular, trimethoprim had a faster rate of passage only in Caco 2 cells and in the BL to AP direction, while D-cycloserine was exclusively transported by Caco 2 cells in the AP to BL direction. In both cases it is possible that active transport mechanisms are involved.