Effect Of Copper On Tightjunctions Permeability

3.1. Introduction

The effect of copper on epithelial permeability was investigated by our group using the Caco-2 cell line (49). In ref. 49, CuSO4 was added either to the AP or to the BL compartment at pH 7.4 in complete medium for 24 h. Under these conditions, copper was chelated by amino acids and proteins (e.g., albumin) present in the complete medium and an increase in monolayer permeability was only observed after treatment with 100 pM CuSO4 from the BL side, which resulted in cellular damage, as shown by a decrease in total protein content (49). Further studies demonstrated that ionic Cu(II), added to the AP side of filter-grown differentiated Caco-2 cells in saline solution at pH 6.0 and at pH 7.0, decreased the monolayer permeability in a dose-dependent way from 20 to 100 pM after 3 h of treatment, but only at pH 6.0 (50). These results demonstrate that copper speciation can play a crucial role in modifying intestinal permeability.

When ingested with food, copper is complexed with low-molecular-weight ligands such as amino acids and organic acids, which can influence its bioavailability. Moreover, copper can be given as a nutritional supplement, both as a salt or as a complex with organic molecules (51). However, the stability of such complexes should be assessed after gastric transit at low pH and the gradual increase in pH occurring in the small intestine. Ionic copper can also be present in drinking water as a result of contamination from wells or pipes, especially in slightly acidic conditions.

In order to investigate the effect of different chemical species of copper on intestinal permeability Caco-2 cells were treated with CuCl2, Cu(His)2, and Cu(Gly)2 (chelazome™).

3.2. Materials and Methods

To investigate the effects of copper on the permeability of tight junctions, Caco-2 cells were treated for 3 h at 37°C with increasing concentrations of CuCl2 in HBSS containing 10 mM morpholinoethane sulfonic acid (MES) (pH 6.0) in the AP compartment. The BL compartment contained HBSS additioned with 10 mM HEPES at pH 7.4, 0.4 % copper-free bovine serum albumin (BSA), and 120 pM reduced glutathione. The BSA had previously been extensively dialyzed against 0.2 M acetate buffer, pH 5.0, to remove copper.

Experiments were 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.

The Cu(His)2 complex was freshly prepared by dropwise addition of 2 mM CuCl2 in HBSS under continuous vortexing to 4 mM L-histidine in HBSS at pH 6.0. The complex was then diluted in HBSS to the required concentration. The copper Chelazome amino acid chelate 2.5% copper complex [Cu(Gly)2], provided by Albion Laboratories (Clearfield, UT), was dissolved in HBSS at the required concentration at pH 6.0 and used fresh.

At the end of copper treatment, the cell monolayer was washed with HBSS and the permeability of the tight junctions was determined by measuring the TEER of filter-grown cell monolayers at 37°C in complete buffered culture medium, as described in Section 2.3.1. When TEER was followed during copper treatment, the measurements were taken in HBSS. For TEER measurements, electrodes were pre-equilibrated for 2 h in the appropriate medium (i.e., HBSS or complete medium). The

Fig. 4. The dose dependence of copper effect on tight-junction permeability. Caco-2 cells were treated with increasing concentrations of CuCl2 (5-100 M) in the AP compartment for 3 h at pH 6.0. Following copper treatment, TEER and 3H-mannitol flux were monitored on the same filters. Data are the means ± SD of triplicate filters from a representative experiment.

Fig. 4. The dose dependence of copper effect on tight-junction permeability. Caco-2 cells were treated with increasing concentrations of CuCl2 (5-100 M) in the AP compartment for 3 h at pH 6.0. Following copper treatment, TEER and 3H-mannitol flux were monitored on the same filters. Data are the means ± SD of triplicate filters from a representative experiment.

transepithelial passage of the radiolabeled extracellular marker D-1[3H(N)]-mannitol (specific activity 706.7 GBq/mmol) across the cell monolayer were determined as described in Section 2.3.2.

In recovery experiments, the tight-junction permeability after treatment 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 26 h.

To ascertain if the recovery after copper treatment was dependent on de novo protein or mRNA synthesis, cells were treated with 30 |iM CuCl2 for 3 h prior to transfer into complete buffered culture medium containing 10 |M cycloheximide or 0.25 |g/mL actinomycin D. TEER values were recorded at set time intervals both during copper treatment and during recovery, after copper removal and transfer to buffered complete culture medium at 37°C up to 26 h.

3.3. Results and Discussion

Differentiated Caco-2 cells were treated for 3 h with increasing concentrations of CuCl2 in HBSS at AP (pH 6.0), and TEER and 3H-mannitol permeability were monitored simultaneously. As shown in Fig. 4, the decrease in TEER with increasing concentrations of CuCl2 exhibited a half-maximal dose of approx 20 | M. The increase in mannitol passage started at 20 | M and continued progressively up to 100 |M. These data clearly demonstrate that the relationship between transepithelial flux and TEER is nonlinear. This derives from the fact that, in parallel circuits, components of low resistance can dominate the net resistance, even if present at low frequency. Conversely, flux measurements are essentially the sum of fluxes across all junctional pathways (5). Given these considerations, it can be seen that, particularly at values of several hundred Ohm ■ centimeter squared (as in the case of Caco-2 cell monolayers), very small increments in junctional permeability produced large decreases in TEER (Fig. 4). Conversely, at low TEER values (< 200 Q ■ cm2), relatively large changes in

Fig. 5. Time-course of recovery from copper-induced changes to TEER in Caco-2 cells. Caco-2 cells were treated for 3 h with 30 |lM CuCl2 at pH 6.0 in the AP compartment with or without 0.25 |lg/mL actinomycin D or 10 ||M cycloheximide. TEER was monitored at set time intervals during copper treatment and during the subsequent recovery period in complete culture medium without added copper, up to 26 h. Data from a representative experiment performed in triplicate are shown as means ± SD and are expressed as the percent of their respective control (i.e., no additions, with actinomycin D or with cycloheximide).

Fig. 5. Time-course of recovery from copper-induced changes to TEER in Caco-2 cells. Caco-2 cells were treated for 3 h with 30 |lM CuCl2 at pH 6.0 in the AP compartment with or without 0.25 |lg/mL actinomycin D or 10 ||M cycloheximide. TEER was monitored at set time intervals during copper treatment and during the subsequent recovery period in complete culture medium without added copper, up to 26 h. Data from a representative experiment performed in triplicate are shown as means ± SD and are expressed as the percent of their respective control (i.e., no additions, with actinomycin D or with cycloheximide).

transjunctional flux of hydrophilic solutes is often associated with very modest changes in TEER. Therefore, depending on the degree of alteration of tight-junctional permeability induced by the toxic agent under study, either TEER or transepithelial flux can better describe the effect. In the case of CuCl2 below 50 |M (for 3 h), TEER modifications are more sensitive indicators of the damage, whereas above this concentration, changes in mannitol flux probably discriminate better the alteration in permeability.

As previously shown, the effect of CuCl2 on the permeability of tight junctions in Caco-2 cells reached a maximum at AP, pH 6.0, and was closely correlated with the AP uptake of copper (40,50). In addition, no damage to cell membranes after treatment with up to 100 | M CuCl2 for 3 h was detected by lactate dehydrogenase (LDH) release assay (40).

To determine whether the effects of copper on tight junctions were reversible, after 3 h of treatment with 30 |M CuCl2, exogenous copper was removed and the cells were transferred to complete buffered medium at 37°C and the TEER values were monitored at regular time intervals for up to 25 h. As shown in Fig. 5, upon copper removal, the TEER, after an initial lag, slowly started to increase, reaching control values after approx 22 h. The addition of either 0.25 |g/mL actinomycin D or 10 |M cycloheximide during the period of recovery indicated that mRNA or protein synthesis inhibitors completely prevented the recovery from occurring after copper removal (Fig. 5).

Reversibility of toxic effects to tight-junction permeability has previously been reported for cytochalasin B, ethanol, and some absorption enhancers, although in contrast to what observed with copper, in these cases the recovery was fast (within 3-4 h) and did not require mRNA or protein synthesis (52-54). These results, together with the observation that, during recovery, copper did not

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