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

Simonetta Ferruzza1, Maria-Laura Scarino1, Giuseppe Rotilio1,2, Maria Rosa Ciriolo3, Paolo Santaroni1, Andrea Onetti Muda4, and Yula Sambuy1

1 Istituto Nazionale della Nutrizione, 00178 Rome; 2 Dipartimento di Biologia, Università di Roma Tor Vergata, 00133 Rome; 3 Dipartimento di Scienze Biomediche, Università di Chieti, 66013 Chieti; and 4 Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, 00161 Rome, Italy


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 µM CuCl2 (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 64CuCl2, both exhibiting a maximum at pH 6.0. Treatment with CuCl2 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 µM 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.

CuCl2; copper-histidine; uptake; pH; F actin organization; microvilli; junctional proteins; recovery


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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COPPER IS A NUTRITIONALLY essential trace element that plays a fundamental role in the biochemistry of all living organisms. It is involved in the function of several enzymes, such as copper-zinc superoxide dismutase, cytochrome oxidase, lysyl oxidase, dopamine beta -hydroxylase, and ceruloplasmin, and is essential for cellular respiration, free radical defense, neurotransmitter function, connective tissue biosynthesis, and cellular iron metabolism (18). However, like other essential trace elements, copper at certain doses can exert toxic effects (34). Consequently, it is important to establish early signs of toxicity of these molecules to determine adequate and safe dietary recommendations.

Copper is normally present in the diet, both as a natural component and as an environmental contaminant. Moreover, copper is included in several over-the-counter nutritional supplements. Acute copper toxicity is infrequent in humans and is usually a consequence of contamination of foodstuffs or beverages (including drinking water). Furthermore, chronic toxicity in humans has been mainly studied in patients with Wilson's disease, a genetic disorder of copper metabolism, and in patients with infantile cirrhosis in some restricted population groups (28).

The intestinal mucosa is the principal site of copper entry into the body and also the first target for its toxicity. The barrier function of the intestinal mucosa is maintained by the tight junction complex joining adjacent epithelial cells, a highly regulated structure that confers selectivity to the permeability of the small intestine (7, 22). It has been shown that certain toxic substances, in cultured differentiated intestinal cells, produce alterations to tight-junction permeability in the absence of more extensive cytotoxic effects (6, 14, 20, 21, 32). Such alterations are particularly important because, at the level of the intestinal mucosa, an increase in tight-junction permeability can lead to uncontrolled influx of molecules from the lumen and can elicit more widespread systemic toxicity or initiate unwanted immune reactions.

The Caco-2 cell line, derived from a human colon adenocarcinoma, spontaneously differentiates in culture, exhibiting several morphological and functional characteristics of mature enterocytes (29). Caco-2 cells grown and differentiated on permeable filter supports represent a well-established model for the study of intestinal transport and toxicity of nutrients and xenobiotics, including trace elements (1, 13, 31, 32, 36).

In the present work, we have investigated the effects of copper, in the form of CuCl2 or complexed to L-histidine [Cu(His)2], on the permeability of tight junctions in Caco-2 cells and have related these observations to the apical (AP) uptake of copper and to its effects on the actin cytoskeleton, on junctional proteins, and on the ultrastructure of the cell.


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Cell culture. The human intestinal Caco-2 cell line was obtained from Prof. Alan Zweibaum (Institut National de la Santé et de la Recherche Medicale, Villejuif, France). Caco-2 cells were grown and maintained as previously described (9) in DMEM containing 25 mM glucose and 3.7 g/l NaHCO3 and supplemented with 4 mM L-glutamine, 1% nonessential amino acids, 1 × 105 U/l penicillin, 100 µg/l streptomycin, and 10% heat-inactivated FCS (complete culture medium). For experiments monitoring the effects of copper on tight-junction permeability, the cells were seeded on polycarbonate filter cell culture chamber inserts (Transwell, 12 mm diameter, 1.13 cm2 area, 0.45 µm pore diameter; Costar Europe, Badhoevedorp, The Netherlands). For copper uptake experiments, the Transwell filters used were 24 mm in diameter, 4.7 cm2 in area, and 0.45 µm in pore diameter. For F actin localization and immunofluorescence, transparent filters were utilized to allow for microscopy (polyethylene terephthalate track-etched membrane, 25 mm diameter, 4.71 cm2 area, 0.4 µm pore diameter; Becton Dickinson Labware Europe, Meylan Cedex, France). Cells were seeded at a density of 4 × 105 cells/cm2 and were allowed to differentiate for 15-17 days after confluency; the medium was regularly changed three times a week.

Copper treatment. To investigate the effects of copper ions on the permeability of tight junctions, Caco-2 cells were treated for 2-3 h at 37°C with increasing concentrations of CuCl2 in Hanks' balanced salt solution (HBSS) containing (in mM) 137 NaCl, 5.36 KCl, 0.44 KH2PO4, 0.34 Na2PO4, 1 CaCl2, 1 MgCl2, and 5.6 glucose, with 10 mM MES, pH 6.0, unless otherwise stated, in the AP compartment. The basolateral (BL) compartment contained HBSS containing 10 mM HEPES at pH 7.4 with 0.4% copper-free BSA and 120 µM reduced glutathione. The BSA had previously been extensively dialyzed against 0.2 M acetate buffer, pH 5.0.

In experiments describing short-term effects of copper, cells were treated with CuCl2 for 15, 30, and 60 min. At appropriate times, exogenous copper was removed by substituting with fresh HBSS, and cells were maintained in this medium for up to 3 h. Subsequently, recovery cells were transferred in complete culture medium buffered with 10 mM HEPES, pH 7.4 (buffered complete culture medium).

Experiments were normally performed maintaining the medium in the AP compartment at pH 6.0 and that in the BL compartment at pH 7.4; these conditions reproduce the pH gradient existing in vivo across the mucosa of the small intestine. When pH effects on copper toxicity and uptake were investigated, the AP pH was adjusted with 10 mM citrate buffer at pH 4.5, with 10 mM MES in the pH range 5.5-6.5 and with 10 mM MOPS at pH 7.0 and 7.4, whereas the pH in the BL compartment was always maintained at pH 7.4 with 10 mM HEPES.

The Cu(His)2 complex was freshly prepared by dropwise addition of 2 mM CuCl2 in HBSS to 4 mM L-histidine in HBSS under continuous vortexing at pH 6.0. The complex was then diluted in HBSS to the required concentration.

Assessment of tight-junction permeability: transepithelial electrical resistance and mannitol passage. At the end of the experiment, the cell monolayer was washed with HBSS and the permeability of the tight junctions was determined by measuring the transepithelial electrical resistance (TEER) of filter-grown cell monolayers at 37°C in buffered complete culture medium, using a commercial apparatus (Millicell ERS; Millipore, Bedford, MA) as previously described (9). When TEER was followed during copper treatment, the measurements were taken in HBSS. For TEER measurements, electrodes were preequilibrated for 2 h in the appropriate medium (i.e., HBSS or complete medium). TEER was expressed as ohms per square centimeter, after subtracting from the reading the resistance of the supporting filter and multiplying the reading by the surface area of the monolayer. The transepithelial passage of the radiolabeled extracellular marker D-1[3H(N)]mannitol (specific activity 706.7 GBq/mmol) across the cell monolayers was determined as previously described (30). Briefly, 50 µM [3H]mannitol in serum-free culture medium was added to the AP compartment, and, after 1 h of incubation at 37°C, the radioactivity in the BL medium was measured in a liquid scintillation counter (LS1801; Beckman Instruments, Irvine, CA) and the AP to BL mannitol passage was expressed as nanomoles per square centimeter per hour.

In recovery experiments after treatment, the tight-junction permeability was monitored by measuring TEER and the cells were transferred in buffered complete culture medium and maintained at 37°C in the incubator or, for brief periods of time, in a water bath, recording TEER values at set intervals up to 24 h.

To ascertain if the recovery after copper treatment was dependent on de novo protein or mRNA synthesis, cells were treated with 30 µM CuCl2 for 3 h before transfer into complete buffered culture medium containing 10 µM cycloheximide or 0.25 µg/ml actinomycin D. TEER values were recorded at set time intervals up to 26 h.

Lactate dehydrogenase assay. The activity of lactate dehydrogenase (LDH) was determined by a standard ultraviolet spectrophotometric test (Merckotest; Merck, Darmstadt, Germany) using an automated analyzer (DU-70; Beckman Instruments). The LDH activity released in the medium was used as an indicator of membrane damage; the activity was measured in the AP and BL media of Caco-2 cells treated from the AP side with 20-100 µM CuCl2 for 3 h. To obtain total LDH activity, cell monolayers were treated from the AP side with 0.1% Triton X-100 for 30 min and the total activity released was determined. Data for LDH release were expressed as the percentage of the total activity released by the cells of one filter.

64Cu uptake experiments. For uptake experiments, Caco-2 cells were treated for 2-6 min at 37°C with 20 and 60 µM of 64CuCl2 in HBSS in the AP compartment. For each experiment, ~13 mg of ultrapure CuCl2 were irradiated for 3 h in a TRIGA reactor (1 MW) at a flux of around 2.4 × 1012 neutrons/s. The specific activity of the 64Cu used in the uptake experiments was between 1-2 Ci/g of CuCl2.

In the pH experiments, the AP medium was maintained between pH 5.5 and pH 7.0, whereas in the Cu(His)2 experiments the AP medium was kept at pH 6.0. The BL medium was always maintained at pH 7.4. At the end of the incubation period, the AP and BL medium were collected, and the filters were transferred on ice and rapidly washed three times with 10 mM EDTA in 150 mM NaCl and 10 mM HEPES, pH 7.4, at 4°C. Filters and standards were placed in polyethylene boxes with screw caps, and copper was detected by counting the total 64Cu. The gamma -induced radioactivity of the standards and cells was determined by gamma -spectrometry using a hyperpure germanium crystal at liquid nitrogen temperature and a counting apparatus (Ortec, EG & G, Milan, Italy).

For total protein determination, filter-grown Caco-2 cells were dissolved in 1 mM NaOH and sonicated three times for 5 s at 20 kHz (Vibracell VC100; Sonic and Materials, Danbury, CT) and the protein was assayed by a colorimetric method (19). Differentiated Caco-2 cells on filter had a total protein content of 0.28 ± 0.006 mg/cm2.

Atomic absorption spectroscopy. To monitor the uptake of copper during treatment for 3 h with 30 µM CuCl2 and the intracellular content during 24 h of recovery, samples were analyzed by atomic absorption spectroscopy. The conditions for uptake were the same as described for 64Cu uptake studies but extended over a longer period of time. Recovery was performed as previously described; after washing exogenous copper with 10 mM EDTA in 150 mM NaCl, pH 7.4, at 4°C, cells were dissolved in 1 N NaOH and sonicated as previously described, and an aliquot was kept for total protein determination. Samples were diluted 1:20 in distilled H2O containing HNO3 (final concentration 0.2%) and were read on a Perkin-Elmer 3100 HGA 600 graphite furnace atomic absorption spectrometer (Beaconsfield, UK) using standard conditions and matrix modifiers (10 mg/ml MgNO3 in 0.2% HNO3) specified by the manufacturer (25). All solutions were of ultrapure grade.

Immunofluorescence and fluorescent localization of F actin and nuclei. For immunofluorescence and for F actin localization, cells were seeded on cell culture chamber inserts fitted with transparent membranes and were treated from the AP compartment with 50-300 µM CuCl2 or with 200 µM Cu(His)2 in HBSS, pH 6.0, for 3 h at 37°C and, after rinsing with PBS containing 1 mM CaCl2 and 1 mM MgCl2 (PBS+), were fixed with 2.5% paraformaldehyde in PBS+. Free aldehydes were quenched with 50 mM NH4Cl in PBS+. For immunofluorescent localization of junctional proteins, cells were permeabilized with 0.075% saponin in PBS+ and the cells were treated with primary antibodies and secondary tetramethylrodamine isothiocyanate (TRITC)-conjugated antibodies according to conventional techniques. The following antibodies were used: rabbit polyclonal anti-zonula occludens-1 (ZO-1), rabbit polyclonal anti-occludin, and mouse monoclonal anti-E-cadherin, all supplied by Zymed Laboratories (San Francisco, CA). Secondary TRITC-conjugated affinity purified goat anti-mouse or anti-rabbit IgG were from Cappel, ICN Biomedicals (Opera, Italy). For F actin localization, cells were incubated for 30 min with 1.7 µg/ml phalloidin (1.28 µM) conjugated either with TRITC or with FITC in PBS+ containing 0.075% saponin and 0.2% BSA. After rinsing, filters were mounted in Vectashield (Vector Laboratories, Burlingame, CA). When labeling of nuclei was required, before mounting, the filters were transferred to absolute methanol at -20°C for 3 min and then incubated for 10 min with 50 ng/ml bisbenzimide (H-33258; Boehringer Mannheim Italia, Monza, Italy) in 0.2 M Na2HPO4-0.1 M citrate buffer at pH 5.5.

The cells were viewed with a fluorescent microscope (Axioscope 2; Zeiss, Jena, Germany) or with a confocal laser microscope (Sarastro 2000 CLSM; Molecular Dynamics, Sunnyvale, CA). Vertical and horizontal scans were reconstructed through Image Space software (Molecular Dynamics). In particular, images were collected either with the usual scanning method (i.e., laser scans of subsequent xy planes at various z positions in the specimen) or with cross-sections through data sets along the xz plane (i.e., vertical scans), which provide a direct insight into the axial extension of the scanned structures.

Transmission electron microscopy. One-half of the filters used for F actin localization experiments were fixed for 1 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and postfixed in 1% OsO4 for 30 min. The samples were dehydrated and embedded in Agar 100 resin (Agar Scientific, Stansted, Essex, UK). Ultrathin sections were cut (Ultracut E; Reichert Jung Optische Werke, Vienna, Austria), stained, and observed in the electron microscope (CM 10; Philips, Eindhoven, The Netherlands).

Data presentation and statistical analysis. Data were analyzed with Statview 4.01 software (Abacus Concepts, Berkeley, CA) by one-way ANOVA followed by Scheffé's F test to determine significant differences among the means. All data are expressed as means ± SD.

Materials. All reagents, unless otherwise specified, were from Sigma Aldrich (Milan, Italy). Cell culture reagents and plastic were from Becton Dickinson. D-1[3H(N)]mannitol was supplied by Life Science Products (Brussels, Belgium). 64Cu was prepared at the ENEA Casaccia Research Center (Anguillara, Italy).


    RESULTS
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Differentiated Caco-2 cells were treated for up to 3 h at 37°C with increasing concentrations of CuCl2 at pH 6.0, and the TEER of the cell monolayers was monitored. As shown in Fig. 1A, the cell monolayers showed a time-dependent decrease in TEER values that increased with the concentration of CuCl2. In addition, to determine whether the changes in tight-junction permeability were reversible, after 3 h of treatment the exogenous copper was removed, the cells were returned to buffered complete culture medium at 37°C, and the TEER values were determined after 24 h. After 24 h of recovery, the cells exhibited the following TEER values, expressed as a percentage of control cells: 96.4 ± 4.0% at 10 µM, 95.6 ± 2.9% at 30 µM, and 84.4 ± 4.4% at 100 µM CuCl2 (data are means ± SD of three experiments performed in triplicate).


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Fig. 1.   Time course of copper effects on transepithelial electrical resistance (TEER) in differentiated Caco-2 cells (A) and effect of cycloheximide (Chx) or actinomycin D (Act D) on their reversibility (B). A: time course of TEER changes after addition of increasing concentrations of CuCl2 in Hanks' balanced salt solution (HBSS, pH 6.0) in apical (AP) medium. TEER values were measured in HBSS at 37°C. Data from a representative experiment performed in triplicate are shown as means ± SD. B: after treatment for 3 h with 30 µM CuCl2 in HBSS at pH 6.0 in AP medium, cells were transferred in buffered complete culture medium. TEER was measured at set time intervals (up to 26 h) at 37°C during recovery in untreated cells (control), copper-treated cells (30 µM CuCl2), and in control and copper-treated cells with addition (at arrow) of 10 µM Chx (control + Chx or CuCl2 + Chx) or 0.25 µg/ml Act D (control + Act D or CuCl2 + Act D). Data from representative experiments performed in triplicate are shown as means ± SD.

To further investigate the time course of recovery and its requirements for de novo synthesis of protein or mRNA, cells were treated for 3 h with 30 µM CuCl2 and transferred to complete growth medium supplemented with 10 µM cycloheximide or 0.25 µg/ml actinomycin D. TEER values were monitored for up to 26 h. The initial drop in TEER values on medium change is due to the different conductance in the two media. As shown in Fig. 1B, the TEER values remained unchanged for up to 7 h (from the start of the experiment). Subsequently, the TEER values tended to increase and attained a value identical to control cells by 25 h. However, the presence of mRNA or protein synthesis inhibitors totally abolished the TEER recovery. TEER values of control cells treated with actinomycin D during recovery time were similar to control, whereas cycloheximide only caused a decrease in TEER values after 12 h of treatment.

To test if the decrease in tight-junction permeability was dependent on the length of copper treatment, 30 µM CuCl2 was added to the cells for 15, 30, or 60 min (Fig. 2A). After treatment, the cells were transferred in HBSS up to 3 h. Additionally, cells treated for 3 h with 30 µM CuCl2 were also included, and TEER was measured at set time intervals in all samples during copper treatment and during recovery for up to 25 h.


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Fig. 2.   Effect of copper treatment on TEER decrease and recovery (A) and on intracellular copper content (B) in differentiated Caco-2 cells. A: Caco-2 cells were treated for 15, 30, 60, and 180 min at 37°C with 30 µM CuCl2 in HBSS at pH 6.0 in AP medium. All cells were maintained in HBSS, pH 6.0, up to 180 min, irrespective of length of treatment. Cells were then transferred in buffered complete culture medium (arrow), and TEER was measured at set time intervals (up to 25 h) at 37°C. Data from representative experiments performed in triplicate are shown as means ± SD. B: cells treated with 30 µM CuCl2 in HBSS at pH 6.0 for 15, 30, and 180 min were collected for intracellular copper determination. After copper treatment for 180 min, cells were transferred to buffered complete culture medium (arrow) and collected after 12 h and 24 h. Copper content was determined by atomic absorption spectroscopy. Data from representative experiments performed in triplicate are shown as means ± SD. Inset: data shown in B are expanded from 0 to 180 min, and copper uptake is compared with decrease in TEER (Delta TEER = TEER control - TEER treated, determined at 180 min) observed after short pulses of copper treatment (data from A).

As shown in Fig. 2A, TEER continued to decrease for up to 3 h, even after copper was removed. Furthermore, the extent of TEER decrease was proportional to the time of treatment. An initial lag in TEER recovery was observed, and complete recovery was achieved earlier for cells treated for shorter times than for cells treated for longer times (15 min vs. 180 min).

Intracellular copper content was also measured, and results are shown in Fig. 2B. Up to fivefold increase in copper content was observed for cells treated with 30 µM CuCl2 compared with control cells, but intracellular copper levels remained relatively unaltered during recovery in the absence of added copper. Copper uptake tended to deviate from linearity after 15 min (Fig. 2B, inset). Furthermore, the decrease in TEER (Delta TEER = TEER control - TEER treated, determined at 180 min; see Fig. 2A) and the intracellular copper content exhibited a similar pattern of increase with time (Fig. 2B, inset).

The effects of copper on tight-junction permeability were further investigated by treating Caco-2 cells from the AP side with 20-100 µM CuCl2 in HBSS at pH 6.0 for 3 h, and TEER and[3H]mannitol passage were monitored simultaneously (Fig. 3A). The TEER decrease with increasing concentrations of CuCl2 showed a half-maximal dose of ~20 µM. The increase in the passage of [3H]mannitol became evident at 20 µM and continued to increase progressively up to 100 µM. No change in TEER or mannitol passage was observed when copper was presented to the Caco-2 cell monolayer complexed with L-histidine (Fig. 3A).


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Fig. 3.   CuCl2 and copper-histidine complex [Cu(His)2] have different effects on tight junctions and are taken up by cells at different rates. A: effect of increasing concentrations of CuCl2 (from 0-100 µM) on TEER and on transepithelial passage of 3H-labeled mannitol after 3 h of treatment in AP compartment of differentiated Caco-2 cells. Each point represents mean ± SD from a representative experiment performed in triplicate. B: time course of AP uptake of 60 µM 64CuCl 2 and of 60 µM 64Cu(His)2 in differentiated Caco-2 cells. Data are means ± SD from representative experiments performed in triplicate.

The uptake of 60 µM CuCl2 was investigated and compared with the uptake of 60 µM Cu(His)2 at pH 6.0. As shown in Fig. 3B, the uptake of 60 µM CuCl2 was more or less linear over the first 6 min, whereas no measurable Cu(His)2 was taken up by the cells over the same period of time.

To exclude copper damage to cell membranes, the LDH activity in the AP and BL media was determined after AP treatment with 20-100 µM CuCl2 for 3 h at pH 6.0 and was expressed as the percentage of total LDH activity released by the cells from one filter after Triton X-100 treatment (10,430 ± 580 mU/mg protein). The LDH in the medium of cells treated with up to 100 µM CuCl2 (2,722 ± 1,230 mU/mg protein) after subtraction of the spontaneous LDH release in control cells (2,253 ± 1,199 mU/mg protein) did not exceed 0.05% of the total activity released by the cells after permeabilization with Triton X-100.

The effects of pH on the copper-induced changes to tight-junction permeability and on AP copper uptake are shown in Fig. 4. When Caco-2 cells were treated from the AP side with 20 µM CuCl2 for 2 h at different pH values, ranging from pH 4.5 to pH 7.4, the maximum decrease in TEER values was observed at pH 6.0 (Fig. 4A). The BL pH was always maintained at pH 7.4. Copper uptake was also investigated as a function of pH in the AP compartment between pH 5.5 and pH 7.0. The rate of AP uptake of 20 µM 64CuCl2 in the first 6 min was shown to be significantly higher at pH 6.0 and 6.5 than at pH 5.5 or 7.0 (Fig. 4B).


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Fig. 4.   Effect of pH in AP medium on changes in TEER after treatment of differentiated Caco-2 cells with 20 µM CuCl2 for 2 h in AP medium (A) and on rate of AP uptake of 20 µM 64CuCl2 (B). A: TEER data are means ± SD of 4 experiments (n = 11). Different letters above error bars indicate significant (P < 0.05, one-way ANOVA followed by Scheffé's F test) differences between means. B: rates of uptake of 20 µM 64CuCl2 from AP compartment at different pH values were calculated between 2 and 6 min in linear range of uptake. pH in basolateral (BL) compartment was always kept at pH 7.4. Data are means ± SD of 1 representative experiment (n = 8). Different letters above error bars indicate significant (P < 0.05, one-way ANOVA followed by Scheffé's F test) differences among various pH values.

Because alterations of the permeability of tight junctions are often accompanied by changes at the level of the cytoskeleton, we investigated the effects of copper on the organization of F actin, ZO-1, occludin, and E-cadherin. Caco-2 cells were treated from the AP side for 3 h with 50-300 µM CuCl2, F actin was localized by staining with fluorescent falloidin, and the nuclei were visualized with bisbenzimide (Fig. 5). Figure 5A shows the organization of F actin in control cells, whereas Fig. 5B shows the cell nuclei in the same microscopic field. After treatment with 50 µM CuCl2 there was an overall reduction in F actin staining, with areas of the cell monolayer showing a marked decrease in F actin staining (Fig. 5C). This effect was not a consequence of cell detachment, as the nuclei were still present even in the areas in which F actin was most severely affected (Fig. 5D). At higher concentrations (300 µM CuCl2), the monolayer showed a reduced staining and highly disorganized F actin (Fig. 5E) but an unaltered nuclear staining (Fig. 5F). The tight-junctional proteins ZO-1 and occludin and the adherens-junction protein E-cadherin were also localized by immunofluorescence in cells treated with 300 µM CuCl2 for 3 h and in control cells. As shown in Fig. 6, A and B, the staining for ZO-1 was similar in control and in copper-treated cells. In cells treated with 300 µM CuCl2 (Fig. 6, D and F), the staining for occludin and E-cadherin, albeit more blurred and less well localized to the cell periphery than in control cells (Fig. 6, C and E), was not substantially reduced, whereas F actin staining was highly disorganized and markedly reduced, as shown in Fig. 5E.


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Fig. 5.   Epifluorescence microscopy of F actin localized by tetramethylrhodamine isothiocyanate (TRITC)-phalloidin binding (A, C, and E) and of nuclei stained with bisbenzimide (B, D, and F) in differentiated Caco-2 cells either untreated (A and B) or treated from AP side for 3 h at pH 6.0 with 50 µM (C and D) or with 300 µM CuCl2 (E and F). Control cells display a regular distribution of F actin, with strong staining around cell periphery and in cytoplasm (A), and integrity of cell monolayer is shown by continuous and regularly-spaced carpet of nuclei (B). After treatment with 50 µM CuCl2, some areas in monolayer display a strongly reduced F actin signal (C), although distribution of nuclei appears unaffected (D). Increasing concentration of CuCl2 to 300 µM results in a very extensive disorganization of F actin, with large areas where staining is almost completely abolished (E), whereas there appears to be no cell detachment, as shown by nuclear staining (F). Bars = 20 µm.



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Fig. 6.   Immunofluorescence microscopy of tight-junctional proteins ZO-1 (A and B) and occludin (C and D) and adherens-junction protein E-cadherin (E and F) in control Caco-2 cells (A, C, and E) and in cells treated with 300 µM CuCl2 for 3 h (B, D, and F). ZO-1 staining is similar in control (A) and in copper-treated cells (B). Occludin and E-cadherin staining in copper-treated cells (D and F) appears more diffuse and less tightly associated with cellular borders than in control cells (C and E), although it is not reduced in intensity. Bars = 20 µm.

To better localize the changes in F actin, falloidin-labeled cells were also observed by confocal laser microscopy. As shown in Fig. 7A in a vertical scan of control cells, the F actin signal was prominent near the AP surface and in the microvilli, which appeared as a continuous brush-border-like signal at the top of the cells. F actin was also localized along the lateral surfaces of the cells, delimiting single cells in the monolayer. In the horizontal scan taken just below the AP surface (Fig. 7B), the signal was mostly concentrated in a circumferential bundle around the cells. In cells treated with 50 µM CuCl2, the vertical scan showed a few cells in which the F actin signal was strongly reduced (Fig. 7C); in the corresponding horizontal scan, the areas of the monolayer showing reduced F actin staining appeared composed of a small number of cells (3-5 cells) (Fig. 7D). With increasing CuCl2 concentration, the areas showing reduced and altered F actin staining increased in size and frequency. Figure 7, E (vertical scan) and F (horizontal scan below the AP surface), showed an area of the cell monolayer exhibiting strongly decreased staining for F actin, and the cells all around this patch exhibited a disorganized F actin signal. Conversely, F actin staining in cells treated with 50-200 µM Cu(His)2 was similar to that in untreated cells [Fig. 7, G and H, showing vertical and horizontal scans of cells treated with 200 µM Cu(His)2].


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Fig. 7.   Confocal laser microscopy of F actin localized by TRITC-phalloidin binding in differentiated Caco-2 cells either untreated (A and B) or treated from AP side with increasing concentrations of CuCl2 (C-F) or Cu(His)2 (G and H) for 3 h. Arrowheads indicate where vertical and horizontal scans were taken. A, C, E, and G are vertical scans, and B, D, F, and H are corresponding horizontal scans taken at level of arrowheads. F actin signal in a vertical scan of control cells (A) appears regularly distributed and continuous, delimiting AP surface and microvilli of brush border; all cells also show signal along lateral membrane. In corresponding horizontal scan of control cells (B), F actin signal is uniformly distributed, delimiting periphery of all cells and, in some areas, is more intense and is also localized in cytoplasm, indicating subcortical cytoskeleton. This apparently irregular distribution of F actin results from different planes in each cell at which horizontal scan was taken. In vertical scan of Caco-2 cells treated with 50 µM CuCl2 (C), AP and later F actin signal completely disappears in an area of monolayer that appears negative for signal also in corresponding horizontal scan (D). Caco-2 cells treated with 200 µM CuCl2 show an uneven and strongly reduced staining both at level of AP surface and along lateral membrane that is evident in vertical (E) and in horizontal (F) scans. Treatment with 200 µM Cu(His)2 (G and H) does not alter F actin distribution compared with control cells. Bars = 20 µm.

Ultrastructural examination of control and copper-treated Caco-2 cells by transmission electron microscopy is shown in Fig. 8. Control cells (Fig. 8, A and B) had normal-appearing microvilli that were homogeneous in size and regularly distributed along the plasma membrane. Also, tight-junction complexes and desmosomes displayed a normal structure and proper organization. With increasing concentrations of copper treatment, a number of ultrastructural alterations were observed. These ranged from disorganization of the microvilli, with normal appearing tight junctions and desmosomes (Fig. 8, C and D), to vast detachment and ballooning of microvilli (Fig. 8E), opening of junctional complexes, and disappearance of desmosomes (Fig. 8F).


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Fig. 8.   Transmission electron microscopic photographs of differentiated Caco-2 cells. Control specimens display well-organized microvilli (A) and normal junctional complexes (B). After treatment with 50 µM CuCl2 for 3 h, microvilli frequently lose their regular organization and are no longer perpendicular to cell membrane (C); however, in such areas, tight junctions and desmosomes appear still preserved (D). After treatment with 200 µM CuCl2, shape and structure of microvilli are hampered; in severely affected areas, microvilli appear completely disorganized, at times detached and often blown up (E). In such cells, tight junctions are clearly open and desmosomes are not evident (F). Arrows indicate tight junctions, and arrowheads indicate desmosomes. Bars = 100 nm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Differentiated Caco-2 cells treated from the AP side with CuCl2 at pH 6.0 showed alterations in the permeability of tight junctions. These copper-induced changes were both time and dose dependent, as monitored by a decrease in TEER and an increase in transepithelial permeability to mannitol. Copper is known to be an efficient producer of reactive oxygen species, often leading to membrane damage. However, there was no indication of membrane damage under our experimental conditions, as shown by the lack of LDH release after CuCl2 treatment. Indeed, we have previously shown (10) that different antioxidants (ascorbic acid, vitamin E, mannitol, dimethylsulfoxide) were unable to counteract the effects of CuCl2 on tight junctions.

The effect of copper on TEER is reversible within 24 h after treatment for 3 h with up to 100 µM CuCl2. Reversibility of the effects of toxic agents on tight-junction permeability in Caco-2 cells have previously been reported for cytochalasin B, some absorption enhancers, and ethanol, although in these cases recovery was rapid (within 3-4 h) and did not require mRNA or protein synthesis (20, 21, 33). The time course of recovery after copper treatment was much slower, with a lag of several hours, and was inhibited by cycloheximide and actinomycin D, suggesting a requirement for mRNA transcription and subsequent protein synthesis for restoration of tight-junction functionality. The full effect of copper on TEER was observed after 3 h of exposure. However, after briefer pulses of copper treatment, the TEER continued to decrease for up to 2-3 h before reaching a plateau value that was maintained for several hours before the onset of the prolonged recovery. Furthermore, the copper effect on TEER closely parallels copper uptake. On the other hand, the onset of recovery was not accompanied by a reduction in intracellular copper, suggesting that, rather than efflux out of the cell, copper undergoes intracellular redistribution from its initial target to other binding proteins, such as metallothionein (5). The long lag before the start of recovery may, at least in part, be due to the need for transcriptional induction of metallothionein. In Caco-2 cells, metallothionein mRNA induction was observed after 5 h of exposure to copper (35). Other intracellular ligands of copper that may participate in this redistribution of the metal include GSH and copper-zinc superoxide dismutase (4).

The transepithelial permeability of tight junctions can be determined by measuring TEER or paracellular flux of soluble tracers such as [3H]mannitol. Neither TEER nor paracellular flux, however, depends only on junctional permeability, and, because they reflect different functional properties, they do not necessarily develop in parallel (22, 23). Indeed, at low copper concentrations, a large decrease in TEER corresponded to a small increase in mannitol permeability, whereas at higher copper concentrations the decrease in TEER slowly leveled off and the permeability to mannitol continued to rise (Fig. 3A). It is important to note that TEER has to decrease below a certain value before molecules such as mannitol can get through the tight junctions (3).

Copper in the intestinal lumen can form coordination bonds with several other molecules originating from the diet. Although amino acids have been suggested to be mandatory ligands for the intestinal uptake of copper, the proportion of ligand to metal is likely to determine whether there is a positive or negative effect of copper chelators on absorption (39). In particular, Cu(His)2 has a positive role in the absorption of copper by liver cells (24), but negative effects on its absorption by the intestine have been reported (39). In Caco-2 cells, Cu(His)2 did not affect the permeability of tight junctions at concentrations in which CuCl2 induced a dramatic increase in tight-junction permeability. Accordingly, the AP uptake of 60 µM 64CuCl2 was much higher than that of 64Cu(His)2 measured over the first 6 min of linear uptake, pointing to a link, in Caco-2 cells, between copper uptake and its effects on tight junctions.

The pH gradient between the AP and BL compartments reproduces the pH conditions of the microenvironment in the proximity of the small intestinal villi and in the submucosal compartment, which provides the driving force for the transport of several nutrients (17, 37). Another proton-dependent transporter that is highly expressed in the small intestine is the divalent cation transporter DCT-1 that, among other cations, has been shown to transport copper (12). The AP uptake of CuCl2 was therefore investigated in Caco-2 cells by varying the AP pH from pH 5.5 to pH 7.0 while the BL pH was maintained at pH 7.4. The maximum uptake of copper at AP pH 6.0-6.5 may partially be explained with the operation of a proton-dependent uptake mechanism, although the presence of other mechanisms of copper internalization cannot be excluded. When the effect of CuCl2 on TEER was assayed after 2 h of treatment at different AP pH values, the maximal effect was observed around pH 6.0. Overall, although the characteristics of apical copper uptake in Caco-2 cells need to be investigated in more detail, our results are compatible with the operation of a copper transporter on the AP surface functioning around a value of pH 6.0, resulting in an intracellular accumulation of copper that leads to alterations in tight-junction permeability.

A further indication that copper effects on tight junctions depend on intracellular events is that F actin distribution was markedly changed after AP treatment of Caco-2 cell monolayers with CuCl2 at pH 6.0. On the other hand, cells exhibiting heavily perturbed F actin showed only limited effects on the localization of the tight junction (ZO-1 and occludin) and adherens-junction (E-cadherin) proteins. The more diffuse localization of occludin and E-cadherin around the cellular borders may result from the extensive disorganization of the F actin cytoskeleton in copper-treated cells.

The ultrastructure of the microvilli appeared markedly altered by the copper treatment. Similar changes in microvilli morphology, appearing distorted and dilated, have previously been observed in kidney proximal tubule epithelium (27) and in isolated rat hepatocytes (16) in which F actin depolymerization had been induced by ATP depletion or by changes in intracellular calcium. Alterations in the morphology of microvilli in liver cells of copper-loaded animals have also been reported, although the mechanisms involved were not investigated (11). The F actin cytoskeleton that supports the AP microvilli is also linked to the tight junctions; immediately below the tight junctions, the adherens junctions are tightly coupled to a circumferential actin-myosin II ring that is a dynamic structure that may transmit cytoskeletal changes to the junctional complex, thus altering tight-junction permeability (26). Drugs that disrupt the actin cytoskeleton, such as cytochalasin, also perturb the paracellular barrier, probably through effects on perijunctional actin (7, 20). Similarly, several agents that alter the permeability of tight junctions, including xenobiotics and natural toxins, have been shown to induce depolymerization of F actin (6, 14, 21, 22) and perturbation of tight-junctional proteins (20, 21). In addition, molecules involved in intracellular signaling pathways, including tyrosine kinases, calcium, protein kinase C, heterotrimeric G proteins, calmodulin, cAMP, lipid second messengers, and phospholipase C, have been reported to affect tight-junction permeability, and their effects are often correlated with changes in actin organization (26).

Copper has previously been reported to induce depolymerization and disorganization of cortical F actin in Mytilus galloprovincialis hemocytes (8). Copper is known to have a high-affinity binding site on a COOH-terminal cysteine residue (Cys-374) of actin, although it has recently been reported that this binding does not affect its state of polymerization (15). However, the effects of copper on the F actin cytoskeleton may not be direct and could be mediated by cytosolic factors such as actin-associated proteins, as recently shown for cadmium in renal mesangial cells (38).

The perturbation of F actin observed in the present work was localized to focal areas, indicating that not all cells in the monolayer were affected to the same extent. However, Caco-2 cells are well known to be a heterogeneous cell population (3), and differences in the levels of copper uptake throughout the monolayer may be expected.

In conclusion, we have shown that ionic copper is able to increase tight-junction permeability in differentiated human Caco-2 cells probably through an intracellular mechanism that involves perturbation of the F actin cytoskeleton. The physiological significance of these observations may be related to the role of copper, a dietary component but also a possible food contaminant, in the alteration of intestinal mucosal permeability. Continuous challenge of intestinal cells from copper and other agents able to perturb the actin cytoskeleton, thus affecting tight-junction permeability, may have more widespread effects at the systemic level. Opening of tight junctions, allowing indiscriminate flow of copper to the circulation, does in fact overcome the innate cellular defense against excess trace element ingestion (such as metallothionein induction, intracellular reduced glutathione levels, superoxide dismutase activity, and so forth). This can in turn lead to systemic toxicity in organs that accumulate copper (i.e., the liver) (28). In addition, small but repeated increases in tight-junction permeability induced by food contaminants such as copper or toxins may have a role in the ever-increasing incidence of food allergies, especially in infants (2).


    ACKNOWLEDGEMENTS

We thank G. Capannesi for preparation of 64Cu, F. Nobili for assistance in elaboration of the figures, A. Di Giacinto for preparation of transmission electron microscopy specimens, and L. Virgili for photographic work. We are grateful to H. J. McArdle for copper analysis and to K. Islam for critical revision of the manuscript.


    FOOTNOTES

This work was supported by European Community FOODCUE Contract no. FAIR-CT95-0813.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M.-L. Scarino, Istituto Nazionale della Nutrizione, Via Ardeatina 546, 00178 Rome, Italy (E-mail: deneb{at}inn.ingrm.it).

Received 21 September 1998; accepted in final form 27 August 1999.


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DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(6):G1138-G1148
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