The Chemical Species of Aluminum Influences Its Paracellular Flux across and Uptake into Caco-2 Cells, a Model of Gastrointestinal Absorption

Yuzhao Zhou* and Robert A. Yokel*,{dagger},1

* Graduate Center for Toxicology, University of Kentucky Medical Center, Lexington, Kentucky 40536–0305; {dagger} College of Pharmacy, University of Kentucky Medical Center, Lexington, Kentucky 40536–0082

1 To whom correspondence should be addressed at 511C Pharmacy Building, University of Kentucky, 725 Rose Street, Lexington, KY 40536-0082. Fax: (859) 323–6886. E-mail: ryokel{at}email.uky.edu.

Received January 25, 2005; accepted May 31, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aluminum (Al) can cause neurotoxicity, a low-turnover osteomalacia, and microcytic anemia. To test the null hypothesis that the chemical form (species) of Al does not influence its mechanism or rate of absorption from the gastrointestinal tract, Al flux across and uptake into Caco-2 cells was investigated. Caco-2 cells were grown on porous membranes mounted in vertical diffusion chambers or in 35-mm-diameter plastic cell culture dishes. When 8 mM 27Al was introduced as the ion, citrate, maltolate, fluoride, or hydroxide, the apical to basolateral apparent permeability (Papp) of Al correlated highly with the Papp of lucifer yellow (LY), a paracellular marker, except when introduced as Al hydroxide. The uptake rate of Al when introduced as the fluoride was > when introduced as the ion > maltolate > citrate > hydroxide. The activation energy of Al introduced as the ion, citrate, maltolate, and fluoride, determined from Årrhenius plots, was 13–22 KJ/mol, suggesting diffusion-mediated uptake. With exposure to 2 µM Al (containing 26Al as a tracer) introduced as the ion, hydroxide, citrate, and fluoride, Al and LY Papp were consistent with results obtained with 8 mM Al, but were not Al species dependent. Approximately 0.015% of the 26Al fluxed across the cell monolayer; 0.75% was associated with cells. Lumogallion staining imaged by confocal laser microscopy showed Al co-localized with DAPI in the nucleus. The results suggest that (1) soluble Al species predominantly diffuse through the paracellular pathway, (2) the ligand-dependent flux rate of Al is due to an effect on the tight junctions, (3) Caco-2 cell uptake of Al is a diffusion process, and (4) the ligand can influence the rate of cellular Al uptake.

Key Words: aluminum absorption; Caco-2 cells; chemical species; flux; paracellular pathway and uptake.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aluminum (Al) has no demonstrated essential function in mammals. Evidence suggests it plays a causal role in the encephalopathy, osteomalacia, and microcytic anemia of chronic renal failure. The controversy surrounding its contribution to Alzheimer's disease has not been resolved (Yokel, 2004Go).

Major sources of Al intake include food, drinking water, and medications (Yokel and McNamara, 2001Go). Daily dietary Al intake is 1–10 mg. Its use as an antacid and/or phosphate binder results in much greater ingestion. Oral Al bioavailability is <1% (Yokel and McNamara, 2001Go). This very low absorption may be due to formation of insoluble Al species at neutral pH (Reiber et al., 1995Go).

The chemical species of Al has been shown to influence its oral absorption. Co-ingestion of silicon appears to reduce Al absorption by formation of insoluble aluminosilicates (Edwardson et al., 1993Go). Citrate enhancement of Al bioavailability has been shown in many studies (e.g., Schönholzer et al., 1997Go; Yokel and McNamara, 1988Go). Two likely mechanisms are (1) formation of an Al-organic acid complex that increases Al solubility and (2) opening of the tight junction (TJ) between cells by citrate to facilitate Al absorption through the paracellular pathway (Froment et al., 1989bGo; Partridge et al., 1992Go).

Fluoride, 0.5 to 1 ppm (mg/l), is frequently added to drinking water to prevent dental caries. Aluminum and fluoride can form stable complexes (Martin, 1988Go). Addition of fluoride increased plasma Al concentrations in rats compared to Al alone (Allain et al., 1996Go). Fluoride has been reported to increase Al-induced neurotoxicity (Stevens et al., 1987Go). Addition of 0.5, 5, or 50 ppm (0.0185, 0.185, or 1.85 mM) Al fluoride to the drinking water of rats for 45 weeks decreased hippocampal cell number and increased brain Al concentration, compared to drinking water with no added Al fluoride (Varner et al., 1993Go).

The oral bioavailability of Al, dosed as maltolate, was reported in one study that was apparently based on a small number of rats (Schönholzer et al., 1997Go). That study found 0.1% oral Al bioavailability, comparable to Al hydroxide, but much less than from Al citrate (1 to 5%). A study was proposed to determine the neurotoxic endpoint of Al consumed in drinking water using Al maltolate (Health Canada, 2001Go). Al maltolate was selected to maintain Al in solution at neutral pH. The results of such a study may not be readily accepted because of the absence of maltolate in drinking water, unless it is demonstrated that the chemical species of Al has little effect on its absorption from the gastrointestinal tract.

Caco-2 cells, derived from the human lower intestine, are a widely used model of the intestinal tract to study rates and mechanisms of oral absorption. They exhibit many features of small intestinal cells, including formation of a highly polarized monolayer. They have been used to study transport of cadmium, calcium, copper, iron, and zinc. The only published study using Caco-2 cells to investigate Al found similar flux when Al was introduced as the nitrilotriacetate or citrate, which was much more rapid than the lactate (Alvarez-Hernandez et al., 1994Go).

The aims of the present studies were to investigate the influence of the chemical species of Al on its distribution across and uptake into Caco-2 cells. The results should greatly enhance understanding of the importance of the chemical species of Al on its oral bioavailability and aid in the risk assessment of Al toxicity from various species. Aluminum, introduced as the ion, hydroxide, citrate, maltolate, and fluoride, was studied. Aluminum species selection was based on the degree of enhanced Al absorption by the associated ligand, the toxicity of some Al species, and the need to assess bioavailability equivalence for representative Al species. Both 2 µM and 8 mM Al were investigated, with 26Al as a tracer and 27Al, respectively. These concentrations correspond to the typical Al concentration of drinking water and those achieved in the gastrointestinal tract during some medical uses of Al. For example, many antacids have 300 to 600 mg Al hydroxide (~104 to 208 mg Al; 3.8 to 7.7 mmoles Al) per tablet/capsule/5 ml. Because of the ubiquitous environmental and biological presence but very low oral bioavailability of 27Al, the use of 26Al with its quantification by accelerator mass spectrometry (AMS) is the best way to study Al toxicokinetics at concentrations relevant to drinking water. However the cost of 26Al and its analysis by AMS is high, limiting its research utility. Therefore, most of the present studies were conducted with 27Al.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Caco-2 cells were obtained from the American Type Culture Collection at passage 17. Aluminum citrate was purchased from City Chemical LLC. Aluminum maltolate was purchased from Gelest Zac or prepared as described elsewhere (Finnegan et al., 1986Go). The former was found by nuclear magnetic resonance (NMR) and the latter by NMR and mass spectroscopy (MS) to have >98% purity. Cell culture reagents were purchased from GIBCO, and cell culture plastic flasks and dishes were purchased from Fisher. 26Al (15 Ci/mmol) was obtained from the Purdue Rare Isotope Measurement Lab (PRIME Lab). Lumogallion was purchased from MPI Research. 3H-propranolol (28 Ci/mmol), DAPI, lucifer yellow, rhodamine 123, and all other chemicals were obtained from Sigma.

Cell culture methods.
Caco-2 cells were routinely grown in 75 cm2 flasks in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% minimum essential medium (MEM) nonessential amino acids, 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin. They were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were passaged at a split ratio of 1:3 in a trypsin-ethylene diamine tetraacetic acid (EDTA) solution at 80% confluence.

Flux studies.
Caco-2 cells were seeded, at passages 20 to 37, on Snapwell polycarbonate 12-mm-diameter, 0.4-µm pore size filters (Corning Costar Corp.) with a seeding density of 105/filter. The medium was changed every other day and 24 h before a flux experiment. Hanks' balanced salt solution, modified by removing phosphate, was used as the flux medium, at 37°C, unless noted otherwise. It contained, in mM: Na+ 142.2, K+ 4, Mg2+ 0.77, Ca2+ 1.25, Cl 145.5, 4.2, 0.27, and D-glucose 5 at pH 7.4, 6.2, or 4 and was isotonic.

Cells were used for the flux study 21 to 28 days post-seeding. Snapwells were mounted in a NaviCyte Vertical Ussing/Diffusion Chamber (Harvard Apparatus). The tightness of the Caco-2 monolayer was measuring as transepithelial electrical resistance (TEER) across the Caco-2 cell monolayer using a RMA3121-Millicell-ERS voltohmmeter (Millipore Corp.). The TEER at the beginning of these studies was ≥350 {Omega} · cm2, indicating an intact monolayer; TEER values were also obtained after completion of flux experiments at 2 h.

To verify that the Caco-2 cells were expressing carriers at the time of their study, P-glycoprotein (P-gp) activity was assessed by measuring basolateral to apical (B to A), rather than apical to basolateral (A to B), transport. Uptake of the P-gp substrate rhodamine 123 (50 µM) was studied in the presence and absence of the P-gp inhibitor verapamil (200 µM).

To monitor flux through the paracellular pathway, lucifer yellow (LY, 100 mg/l), which fluxes across cells via this pathway (Hidalgo et al., 1989Go), was added to the medium on the donor side of the cell monolayer of all studies. To assess transcellular flux, propranolol (100 µM), which fluxes transcellularly across cells (Walgren et al., 1998Go), was added to the donor medium in some studies at pH 7.4. Samples of the medium from the donor chamber were collected at the start, and from the receiving chamber at the end, of the experiment for analysis of these markers.

27Al was introduced into the donor chamber medium, usually on the apical side of the Caco-2 cell monolayers, as the Al ion at pH 4, Al hydroxide at pH 6.2, Al citrate at pH 7.4, Al maltolate at pH 7.4, or Al fluoride at pH 4, and compared to studies in which no Al or only the associated ligand (citrate, maltolate, or fluoride) was added. The generation of these Al species is described below. Initial studies were conducted for both A to B and B to A flux with 0.5, 2, 8, and 50 mM Al, as the citrate in the presence or absence of Ca in the medium. Samples (150 µl) were taken from the donor chamber at time 0 and from the receiving chamber after 15, 30, 45, 60, 90, and 120 min. Results showed that (1) Ca is required to maintain the TJ of cells; (2) there was no measurable A to B Al flux when 0.5 and 2 mM Al and 1.25 mM Ca were included in the medium; (3) 50 mM Al was toxic. Eight mM Al was found necessary to allow Al determination in the receiving chamber by the analytical method used (below), due to the low flux of Al across these cells. Therefore, 8 mM Al was used in all studies of 27Al flux and uptake. Some experiments with Al ion were conducted in the presence of reduced or no Ca in the medium.

Apical Al Uptake by Caco-2 Cells.
Caco-2 cells were plated into 35-mm-diameter six-well plates (Corning Costar Corp.). Uptake studies were conducted 5 to 10 days after plating. The uptake medium was the same as used for the flux studies, above. Prior to the uptake study, cells were washed three times with uptake medium containing no substrates. One ml of 8 mM Al as the ion, hydroxide, citrate, maltolate, or fluoride was added and incubated at 37°C for up to 4 h. To conduct a temperature-dependence study, uptake was performed at 4°, 12°, 20°, 28°, and 37°C for 30 min. Uptake was terminated by aspiration removal of the uptake medium and washing the cells five times with ice-cold uptake medium containing 5 mM desferrioxamine (DFO), a Al chelator, to remove cell-surface-bound Al. In a preliminary study, we compared Al uptake introduced as the maltolate while washing cells with ice-cold medium containing or not containing DFO. The results showed no significant difference between these two wash procedures and suggested that DFO did not extract Al from inside the cells. To ascertain whether the apparent Al uptake was cell associated or only dish associated, Al solution was incubated in the dish without cells for 30 min at 37°C. The uptake medium was collected for lactate dehydrogenase (LDH) assay, an indicator of cell toxicity. To validate this method, commercial LDH (~10 U/ml) was added to uptake medium, which was assayed and showed UV absorbance. Cells were lysed by 1 ml of 0.1% Triton X-100 for measurement of Al concentration and protein determination. Al uptake after 15 s on ice was determined as a control for nonspecific Al binding to the cell membrane. In selected experiments, the uptake of Al as the ion, citrate, and maltolate was studied beyond 4 h.

Lumogallion staining and confocal microscopic imaging of Al localization.
To determine if Al enters Caco-2 cells or simply associates with the plasma membrane outer surface after Al exposure, we used lumogallion, a stain that is quite selective and sensitive for Al, and fluorescence confocal microscopy to visualize Al localization. The Al–lumogallion complex has a reported limit of detection of 3.6 ng/ml (0.13 µM) (Uchiumi et al., 1998Go). Caco-2 cells were seeded at a density of 1 x 105 cells/cm2 onto 35-mm-diameter glass-bottomed coverslip dishes (MatTeck Corp.) and cultured for 5 to 7 days. The above uptake procedure was used. After 30 min uptake at 37°C, cells were fixed in 1.5 ml 4% paraformaldehyde solution for 15 min at room temperature and then stained in 1 ml acetate buffer (pH 4) containing 2.5 x 10–5 mol/l lumogallion in darkness for 1 h at 50°C. After they were rinsed with acetate buffer twice to remove excess stain, cells were incubated with DAPI (50 µg/ml), a nuclear stain, for 10 min at room temperature. Caco-2 cells not exposed to Al but stained with lumogallion and DAPI served as a negative control. A Leica TCS SP inverted confocal microscope was used to examine the Al location. The excitation wavelength was 488 nm and 364 nm to visualize the Al–lumogallion complex and DAPI stained nuclei, respectively. Emitted fluorescence was collected at wavelengths from 530 to 580 nm for the Al–lumogallion complex and 461 nm for DAPI. The photomultiplier tube (PMT) voltage was selected to produce images of approximately equal fluorescence intensity to generate clear images showing the Al distribution. Differential interference contrast (DIC) images were collected concurrently.

A concurrent flux and uptake study of selected Al species using 26Al as an Al tracer.
The objective was to determine if the above observations, obtained with exposure to 8 mM Al, generalize to much lower Al concentrations. Caco-2 cells grown on Snapwell membranes were mounted in the vertical diffusion chamber, as above. 26Al (~0.15 nCi, ~7.5 ng; total Al ~270 ng), was introduced into the 5 ml of medium in the donor chamber on the apical cell surface, yielding ~55 µg Al/l (2 µM). Aluminum was introduced as the ion, citrate, maltolate, or fluoride to one of the vertical diffusion chambers and compared to no added Al. The experiment was conducted three times. After 120 min, samples of the receiving chamber medium and the cells were taken.

Al species generated under the conditions studied.
The anticipated Al species under the conditions used in these studies were obtained from the literature or were calculated by Dr. Wesley Harris. In the absence of ligands, the solubility of free, unhydrolyzed Al3+ in a pH 4 aqueous solution is 50 mM, based on the solubility constant for Al(OH)3 (Martin, 1992Go). Speciation calculations show that nearly all of the Al in both a 8 mM and 2 µM Al solution at pH 4 would be the Al ion, as shown in Tables 1 and 2 (Harris et al., 1996Go). The nadir of Al solubility is at pH 6.2, allowing <0.05 µM free unhydrolyzed Al3+ ion (W. Harris, personal communication), as a result of the formation of insoluble Al hydroxide (Harris et al., 1996Go). Table 1 shows the Al species at pH 6.2 for 8 mM Al, from calculations based on the constants used in Harris et al. (1996)Go. The Al citrate species shown in Tables 1 and 2 resulted from calculations for 8 mM and 2 µM Al and equimolar citrate at pH 7.4, based on constants reported by Lakatos et al. (2001)Go. Aluminum fluoride was prepared by adding AlCl3 and sodium fluoride to the uptake medium and allowing the solution to stand for ≥2 h at room temperature. The calculated species are shown in Tables 1 and 2. When 2 µM Al was introduced in the presence of 6 µM maltol at pH 7.4 the speciation calculations revealed that essentially all the Al was Al hydroxide.


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TABLE 1 Al Speciation Calculations for a Total [Al] = 8 mM

 

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TABLE 2 Al Speciation Calculations for a Total [Al] = 2 µM

 
Sample analysis.
Aluminum concentration in media and cell lysates was measured by electrothermal atomic absorption spectrometry (ETAAS), with a PerkinElmer 4100 ZL spectrophotometer. An aqueous solution containing 0.2% HNO3 and 2.5 mM Mg2+ was used as the matrix for all Al standards and samples. The limit of detection, determined as blank + 3 SD, was 3 ng Al/ml. Unknowns were compared to aqueous standards containing 5 to 40 ng Al/ml. Each unknown was injected ≥2 times. Results were accepted when the relative standard deviation (RSD) of the replicate injections was <10%. When the Al concentration in background control (medium with no added Al) was below the limit of detection, it was considered to be 0. The Al concentration in all recipient side samples from experiments with added Al was > the limit of detection.

Data analysis

Statistical analysis.
The flux and uptake results versus time were fit to linear or non-linear functions using GraphPad Prism to ascertain the best fit. Correlation coefficient calculations were conducted using GraphPad Prism. One-way analysis of variance (ANOVA) was conducted to compare the effect of the ligands on 26Al flux/uptake and the effect of Al species versus control on changes in LY flux and TEER. Statistical significance was accepted at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Al concentration in flux and uptake media in the absence of added Al (background Al contamination) was <4 ng/ml.

Flux Studies
The B to A permeability of rhodamine 123 was 5.58 ± 0.66 x 10–6 cm/s, consistent with a reported value (1.62 x 10–5 cm/s) (Troutman and Thakker, 2003Go). Addition of 200 µM verapamil inhibited B to A flux and increased rhodamine 123 uptake. The results show the Caco-2 cells expressed P-gp under the conditions that were used.

The time course of Al flux from the A to B side of the Caco-2 cells after introduction of the five Al species ([Al] = 8 mM ) is shown in Figure 1. Flux of Al, as the ion and hydroxide, was best fit by a linear function. Flux of Al as the citrate, maltolate, and fluoride was best fit by a non-linear function. There was indication of a ~30 min lag time in the time course of Al species flux. The flux for Al in the presence of fluoride dramatically increased at 2 h, which correlated with a decreased TEER, indicating the opening of the TJ (paracellular pathway) between cells. Transepithelial electrical resistance generally decreased ~25% during the 2-h flux experiment in the absence of added Al (Fig. 2). For most Al species, this was not significantly different from control, whereas after 2-h exposure to 8 mM Al in the presence of fluoride, or 32 mM fluoride alone (not shown), the decrease in TEER was significant.



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FIG. 1. The time course of apical to basolateral flux of the five aluminum species across Caco-2 cells. The cells were exposed to 8 mM Al. Results are a representative observation from at least three experiments, each conducted with two replicates, for each Al species. Note change in Y axis scale with Al fluoride.

 


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FIG. 2. The decrease in transepithelial electrical resistance (TEER) across Caco-2 cells during 2 h of flux of the five Al species studied. The cells were exposed to 8 mM Al. Values shown are mean ± SD as a percentage of the TEER at the start of the experiments. *p < 0.05 versus control.

 
The fluxes of LY and propranolol across the Caco-2 monolayer were determined concurrently. The Papp of propranolol was 24.3 ± 1.9 x 10–6 cm/s, consistent with a reported value (23.4 ± 2.8 x 10–6 cm/s) (Walgren et al., 1998Go). Across the experiments where the Papp of LY increased from 0.18 to 1.8 x 10–6 cm/s, the Papp of propranolol did not change (results not shown).

The Papp of Al hydroxide was the lowest of the five Al species studied (Fig. 3). Cloudiness was observed in this condition where there was no added ligand to bind Al. The Papp of Al as the ion, citrate, maltolate, and fluoride positively correlated with the Papp of LY, whereas the Papp of Al hydroxide did not significantly correlate with the Papp of LY. The Papp of Al flux, as Al citrate, was independent of propranolol flux. In the experiments with reduced Ca in the medium, the Papp of LY and Al increased. A composite of all results of Al flux when 8 mM Al was introduced as the ion, citrate, maltolate, and fluoride, with normal Ca or reduced Ca, shows they are quite close to a single linear relationship (Fig. 4).



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FIG. 3. Apparent apical to basolateral flux, expressed as apparent permeability, of the five Al species compared to lucifer yellow (LY). Values shown are flux over 2-h exposure to 8 mM Al in the presence of 1.25 mM Ca, unless noted otherwise. Results are from at least three experiments, each conducted with two replicates. Each point represents one observation. Note change in X and Y axes scales in the Al fluoride panel. The correlation coefficients are 0.89, 0.49, 0.98, 0.99, and 0.99 for the Al as the ion, hydroxide, citrate, maltolate, and fluoride, respectively.

 


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FIG. 4. Apparent apical to basolateral permeability results from all of the flux experiments. Values shown for the first nine symbols are results obtained with 8 mM Al exposure. The correlation coefficient is 0.99. Values shown for the last four symbols are results obtained with 2 µM Al exposure. The correlation coefficient for all results shown is 0.99.

 
The B to A permeability of both Al and LY were greater than A to B when 8 mM Al citrate was added to the donor medium. The time course of Al flux from B to A was best fit by a linear function (data not shown).

Studies were conducted to assess the effect of citrate, maltolate, and fluoride alone. Each was tested in two to three experiments with a single replicate. Only fluoride decreased the TEER and increased LY permeability, indicating toxicity from this ligand.

Apical Al Uptake by Caco-2 Cells
The uptake kinetics of each of the five Al species was different from the other four (Fig. 5). The time course of Al uptake as the hydroxide looked like adsorption because its uptake was less than 2 times the non-specific Al binding to the cells, e.g., the Al associated with the cells after 15 s on ice. The uptake of Al as the other species was at least 5 times higher than the nonspecific binding. The Al associated with the cells after 15 s on ice was 0.85, 6.19, 0.04, 0.04, and 2.82, as a percentage of the Al introduced/mg cell protein, for Al introduced as the ion, hydroxide, citrate, maltolate, and fluoride, respectively. The kinetics of Al ion and Al maltolate were best fit by a linear function. Aluminum citrate kinetics were best fit by a two-phase exponential association. There appears to be a rapid initial uptake of Al, when introduced as the ion, citrate, maltolate, and hydroxide. The uptake rate of Al as the ion was greater than for Al as the maltolate and citrate. Aluminum in the presence of fluoride exhibited the highest uptake rate of the five Al species. However, after 120-min and 240-min exposure to 8 mM Al fluoride, the Al associated with the cells decreased. The LDH assay indicated cell toxicity at these time points (results not shown). LDH release in the absence of added Al at pH 4 after 240 min was not significantly different from control, indicating that the effects seen with Al fluoride were not due to this pH.



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FIG. 5. Time course of uptake of the Al species into Caco-2 cells. Upper panel: Results are mean of three replicates in one representative experiment, of at least three experiments conducted for each of the Al species, tested at 8 mM Al. Results are shown together to enable comparison of the five Al species. Lower panels: Results are mean ± SD of four or five experiments each with three replicates for Al citrate (left panel) and Al maltolate (right panel), which were fit to linear or non-linear functions (best fit) using GraphPad Prism. Note the different Y-axis scales in the left and right lower panels to enable visualization of Al citrate and Al maltolate results.

 
Limited uptake experiments were conducted to 8 h with the Al ion and Al citrate and 18 h with Al maltolate. After 2 h exposure to Al fluoride and 18 h exposure to Al maltolate the cells were not healthy, shown by increased LDH release. After 8 h Al citrate exposure the cells became detached. Lactate dehydrogenase release was not increased under the other conditions.

Intracellular Al concentration was estimated based on an intracellular volume of 3.66 µl/mg protein (Blais et al., 1987Go). The results show cellular Al concentrations ~0.7-, 2-, 8-, and 25-fold of the uptake medium, after Al introduction as the citrate, maltolate, ion, and fluoride, respectively, suggesting concentrative uptake of several Al species.

The influence of temperature on Al uptake was determined for Al as the ion, citrate, maltolate, and fluoride. The energies of activation of the four Al species calculated from the Årrhenius plot were not significantly different and were in the range of 13.3–21.6 kJ/mol (3.2–5.2 kcal/mol) (Fig. 6).



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FIG. 6. The temperature dependence of Al uptake (Årrhenius plots) for four Al species. Results are mean ± SD of three experiments, each with three replicates, conducted at 8 mM Al. The results were best fit to a linear function.

 
Confocal Microscopic Imaging of Al Localization
The Caco-2 cell intracellular Al concentration in the uptake experiment was calculated based on cell volume and found to be 37, 392, 741, 8024, 9629, and 12,390 µM for cells not exposed to added Al and cells exposed to Al as the citrate, maltolate, ion, hydroxide, and fluoride, respectively. The intensity of fluorescence was proportional to the intracellular Al concentration. A significant negative correlation existed between the intracellular Al concentration and the PMT voltage used to generate the confocal images of comparable intensity. Images of Al–lumogallion (Fig. 7) showed a similar intracellular Al distribution after exposure to the five Al species and control. Aluminum was not limited to the plasma membrane outer surface. Overlays of the fluorescent images of lumogallion stain for Al and DAPI, with images using DIC to reveal cellular detail, indicate that the Al is localized with DAPI in the nucleus of Caco-2 cells.



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FIG. 7. Confocal microscopic images of Al localization in Caco-2 cells. Left panels: the fluorescent images of DAPI, a nuclear stain. Middle panels: the confocal laser images of lumogallion-stained cells (the voltage of the PMT used was 785, 642, 608, 727, 672, and 602 for control, Al as the ion, hydroxide, citrate, maltolate, and fluoride, respectively). Right panels: overlays of DAPI, lumogallion–Al, and DIC images. Scale bar in right panel Al citrate = 20 µm.

 
Concurrent Flux and Uptake of Al Using 26Al as a Tracer
The average AMS 26Al/27Al ratio from the flux experiments was 3 x 10–10, and from the uptake experiments it was 1.25 x 10–8. The RSD of all samples was <10%. With exposure to 2 µM Al (containing 26Al) as the ion, hydroxide, citrate, and fluoride, the Papp of Al was 1.5 to 16 x 10–8 cm/s while the Papp of LY was 2.8 to 13 x 10–7 cm/s. Aluminum and LY Papp were consistent with results obtained with 8 mM Al (Fig. 4). There were no significant differences in the decrease of TEER (data not shown) or increased Papp of LY among the Al species and control (Fig. 4). Aluminum uptake and flux were not significantly different among the Al species (Fig. 8). Approximately 0.015% of the Al in the uptake medium fluxed across the cell monolayer while ~0.75% was associated with the Caco-2 cells, equivalent to a cellular Al concentration of 5 µm, or a concentration of Al from uptake medium to cell of 2.5.



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FIG. 8. Concurrent flux and uptake of Al (using 26Al as a tracer) by Caco-2 cells exposed to 2 µM Al. Flux and uptake of Al into Caco-2 cells, as a % of the Al introduced on the donor (apical) cell side. Left Y-axis scale is for flux results, right Y-axis scale is for uptake results. Note the difference in the Y-axis scales. Values shown are mean ± SD of three experiments, each conducted with a single replicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this work was to investigate the influence of the chemical species of Al on its intestinal absorption and the mechanism involved, using Caco-2 cells. Most of the studies were conducted using 27Al, quantified by ETAAS, requiring Al concentrations in excess of those seen in drinking water, but relevant to medicinal Al exposure. A better way to address the goals of this work relevant to drinking water might be to use 26Al with AMS quantification, which enables the study of Al at concentrations seen in drinking water. However, the high cost of obtaining 26Al, and more important the high cost of 26Al analysis by AMS, limits its use. Therefore, we initially conducted extensive studies with 27Al to elucidate the processes of Al flux across and uptake in Caco-2 cells and then conducted a smaller study with 26Al and selected endpoints to determine whether the mechanism observed at the higher Al concentration (8 mM) was seen at a lower concentration (2 µM).

Manipulating TJ integrity of Caco-2 monolayers can be used to define absorption routes (i.e., paracellular vs transcellular). The cell lipid bilayer membrane surface area is much larger than the paracellular area. Therefore, the flux of hydrophobic compounds through cells is not sensitive to TJ integrity because the contribution of transcellular flux is much greater than paracellular flux to transepithelial absorption. In contrast, hydrophilic compounds, which flux across Caco-2 monolayers by the paracellular pathway, are very sensitive to manipulations of TJ integrity. Because of their different routes of transmembrane flux, the fluxes of LY and propranolol would not be expected to correlate positively.

In the current study, Al ion and Al fluoride were tested at pH 4 to maintain the desired Al chemical species. Lucifer yellow flux and TEER, in the absence of Al, suggested that this pH had no apparent adverse effect on the integrity of the cell monolayer and paracellular pathway. Similar observations have been reported for pH 4.5, confirmed by TEM (Meaney and O'Driscoll, 1999Go) and pH 4 (Artursson et al., 1994Go). Lactate dehydrogenase release after exposure to uptake medium at pH 4 and pH 7.4 was not significantly different in the absence of Al.

The rates of flux of the five Al species studied were dissimilar, as shown for 2 mM Al introduced as the nitrilotriacetate, citrate, and lactate (Alvarez-Hernandez et al., 1994Go). The present results show a high positive correlation between the flux of Al, as the ion, citrate, maltolate, and fluoride, but not as the hydroxide, compared to LY, suggesting that the four soluble Al species flux across Caco-2 cells through the paracellular pathway. This led to the conclusion that the paracellular pathway was involved in Al absorption, as suggested by studies that used an in situ rat gut preparation (Froment et al., 1989aGo; Partridge et al., 1992Go; Provan and Yokel, 1988Go) and inverted intestinal segments (Farrar et al., 1987Go; Whitehead et al., 1997Go). The site of absorption appears to be the proximal small intestine (Froment et al., 1989bGo; Whitehead et al., 1997Go). The lower permeability of Al hydroxide as compared with the other four species may be due to its limited solubility. The highest permeability of Al was in the presence of fluoride, which was associated with the greatest increase of LY flux and the greatest decrease of TEER, suggesting that fluoride or Al fluoride toxicity to the Caco-2 TJ may have opened the paracellular pathway. Some flux studies were conducted in the absence of Ca or the presence of reduced Ca in the flux medium. Ca2+ is required to maintain the integrity and function of the TJ (Ma et al., 2000Go). In the absence of Ca in the flux medium, the percentage of LY flux from the donor side to the recipient side greatly increased beyond that seen in the presence of 1.25 mM Ca (3.02% vs 0.19%). This was associated with a much higher Al flux across the Caco-2 cell monolayer.

A ~30-min lag time was observed in the time course of Al flux as the citrate and maltolate. This lag time might be the result of the time course of the effect of Al and/or the associated ligand to modulate the tight junction; e.g., an ultrastructure such as the F actin cytoskeleton might be changed. The Al species studied are hydrophilic. Modulating the TJ would permit Al to more readily diffuse between cells.

The B to A flux of Al citrate, and LY in the same experiments, was greater than A to B flux. However LY flux from B to A was not significantly different than from A to B in the absence of Al citrate. The value of the stability constant (K) for the calcium citrate complex is between 6.8 x 104 and 7.1 x 104 l/mol (Singh et al., 1991Go). Aluminum citrate, or citrate, may be better able to open the TJ from the B side by complexing Ca. This is consistent with the ability of basolateral, but not apical, application of ethylene diamine tetraacetic acid (EDTA) to affect TEER (Noach et al., 1993Go). However, we cannot rule out alternative interpretations based on the data provided. For example, Al species might have an effect on the F-actin cytoskeleton to open the tight junction. The basolateral side of Caco-2 cells may be more sensitive to this effect.

The slower uptake rates of Al citrate and Al maltolate than the Al ion may be due to inhibition of Al uptake by these ligands. The ability of ligands, such as citrate, to inhibit Al uptake into plants has been reported (Yokel, 2004Go). An initial high uptake rate was observed for Al introduced as the citrate, maltolate, hydroxide, and ion. Our explanation was this: A large gradient existed between the intracellular Al concentration and medium at the beginning of the Al exposure. The Al concentration gradient drove cell Al entry, and the Al within the cells distributed inside the cells rapidly. Over time the gradient decreased, reducing the uptake rate. The uptake rate of Al in the presence of fluoride was the highest among the five Al species studied. Exposure of the Caco-2 cells to 8 mM Al as the fluoride caused cell toxicity after 120 min (LDH release). This release of a cytosolic enzyme was presumably accompanied by Al release, causing the decreased intracellular Al, as shown in Figure 5. Lactate dehydrogenase release was not increased by pH 4 in the absence of Al fluoride, but it was increased by fluoride alone, suggesting that fluoride and/or Al fluoride were toxic to Caco-2 cells.

The Årrhenius plot can be used to interpret the mechanism of Al uptake. The low activation energies seen in the present study are generally below the range associated with active, energy-dependent transport (Makhey et al., 1998Go), suggesting that Al enters Caco-2 cells through a channel rather than by carrier-mediated transport. Ea (the temperature-dependent phenomenological energy of activation) is generally 6 kcal/mol for water movement through an aqueous pore and 8–10 kcal/mol for water movement through a lipid bilayer (Finkelstein, 1987Go). The low activation energy indicated that the uptake mechanism was close to water diffusion and was probably channel mediated. There are no reports of a carrier for Al on intestinal cells. It is possible that 8 mM Al might saturate carriers, which would only enable us to observe a diffusion process. One way to address this question would be to use 26Al at a much lower concentration, which was done. The 26Al results of our study show concentrative association of Al with Caco-2 cells after introduction of Al as the ion, hydroxide, citrate, and fluoride. These and the confocal results suggest that Al enters Caco-2 cells by a non-energy-dependent process, probably by diffusion through ion channels, and then becomes sequestered intracellularly, e.g., in the nucleus, allowing concentrative Al uptake.

The calculated Al species in the presence of ligands at 8 mM and 2 µM Al are shown in Tables 1 and 2. At 2 µM, the uptake and flux of Al did not show differences among Al species. The very low concentration of ligand-associated Al did not have an effect on the tight junctions. Most Al species are charged, and the integrity of tight junctions presumably limited Al flux between cells under this condition. The four Al species we tested fluxed through the paracellular/transcellular pathways equally. At 8 mM, Al flux appears to be by the paracellular route, facilitated by opening of the tight junctions. We speculated that a high concentration of Al or ligands might change the ultrastructure of cells, e.g., the microvilli or F-actin cytoskeleton. The different ability of the Al species or ligands to modulate the TJ resulted in differing degrees of TJ opening and thereby different flux rates through the TJ. However, results of Al uptake from medium containing 2 µM and 8 mM Al are not directly comparable because of the different time the cells were studied after seeding (5 days and 21 days) and the different support materials (plastic and polycarbonate). The ligand-dependent different Al uptake rates might be due to the effect of ligands on the cell membrane/cytoskeleton and the resulting toxic effect.

Aluminum fluoride has the potential to produce a damaging effect on cell lipid bi-layer membranes and has been shown to influence the transport of some ions such as Na+ and Cl (Suwalsky et al., 2004Go). It has been reported that Gai2 is important for both the maintenance and development of the TJ (Saha et al., 1998Go). Treatment of confluent monolayers of MDCK cells with Al fluoride (3 mm NaF + 50 µM AlCl3, an activator of heterotrimeric G protein a subunits) resulted in a change of transepithelial resistance. A limitation of our study was the use of a high concentration of Al fluoride, which has been reported in the literature to be toxic, potentially influencing our experiments to observe Al flux and uptake. Aluminum showed a very high uptake rate in the presence of fluoride, which may have been caused by a toxic effect on the cell membrane. The increased flux of Al in the presence of fluoride was associated with a decreased TEER. However, in the presence of a low concentration of Al fluoride—e.g., 2 µM in the 26Al study, which did not have an apparent toxic effect—the uptake and flux of Al introduced as the fluoride was not greater than other Al species. Therefore, we cannot generalize the findings obtained with the injured cells to normal healthy cells.

The ability to visualize Al in the control cells, as well as Al-treated cells, can be explained by the high sensitivity of lumogallion, the high voltage used to image these cells, and the influence of probable Al from the environment and reagents. The confocal microscopy images show that Al associated with the Caco-2 cells was not simply adsorbed onto the plasma membrane surface, but that it entered the cells. The intracellular Al localization was heterogeneous with a high Al concentration in the nuclei. Nuclear Al probably binds to nucleotides or phosphorylated proteins (Martin, 1992Go) to disturb cell function. The present study is the first to describe Al localization in Caco-2 cells. Distribution of Al in nuclei, lysosomes, mitochondria, cytoplasm, and associated with cytoskeletal elements has been reported (Dobson et al., 1998Go). Intracellular Al localization may vary, depending on cell type and treatment conditions.

The use of Caco-2 cells provides the opportunity for better control of the chemical species of Al than can be achieved in vivo (in situ). In general, Caco-2 cells are a good model of intestinal absorption and are becoming a standard preparation for predicting the absorption of drugs intended for oral delivery. There is a good correlation between Papp in these cells and oral bioavailability (Yazdanian et al., 1998Go). The permeabilities of polyethylene glycols with molecular weights ranging from 200 to 500, as paraceullular markers, across Caco-2 cells were determined and compared to reported results obtained with human tissue (Artursson et al., 1993Go). The calculated permeability coefficients across Caco-2 cells were approximately 2 and 1 orders of magnitude lower than across human tissue. One study found a TEER of ~470 {Omega} · cm2 for Caco-2 monolayers versus 40 {Omega} · cm2 for rat jejunum (Tanaka et al., 1995Go). Therefore, transport studies of hydrophilic molecules, e.g., ions and paracellular markers, across Caco-2 cells could potentially underestimate transport across intestinal tissue. Although the absolute rates of flux may not translate from in vitro Caco-2 cells in culture to in vivo, it is expected that the relative rates among the Al species studied in Caco-2 cells would generalize to in vivo rates, and that mechanisms of flux across the Caco-2 cell monolayer in vitro would generalize to the mechanisms of flux across Caco-2 cells in vivo. If the paracellular pathway is the predominant route of Al flux across Caco-2 cells, which have tighter junctions than mammalian intestine, it is likely that this pathway, which is less restrictive in the intestine of mammals, also permits Al flux. In the 26Al study, uptake of Al was ~50 times greater than flux across the Caco-2 cell monolayer. However, Al seems to be sequestered in Caco-2 cells (e.g., nuclei), which may prevent significant transcellular Al flux, resulting in Al absorption by paracellular flux. Another limit of Caco-2 cells is that they have a low ability to secrete mucus. The mucus is secreted throughout the intestine and can be a barrier to drug absorption. It has therefore been suggested to play a significant role in Al absorption (Powell et al., 1994Go).

In summary, the present work showed that (1) soluble Al species diffuse through the paracellular pathway of Caco-2 cell monolayers, (2) Caco-2 cell uptake of Al is a diffusion process, (3) the ligand may influence TJ integrity, (4) the ligand can influence the rate of cellular Al uptake, and (5) intracellular Al became localized in the nuclei of Caco-2 cells.


    NOTES
 
This work was presented in part at the 43rd Annual Meeting of the Society of Toxicology, Baltimore, MD, 2004.


    ACKNOWLEDGMENTS
 
This research was supported by grant from the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) program. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through grant/cooperative agreement R-82978301 to R.A.Y., it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. We thank Drs. Patrick J. McNamara and R. Tim Miller, University of Kentucky, for helpful suggestions on the conduct and interpretation of this work, and Dr. Wesley R. Harris, University of Missouri–St. Louis, for many informative discussions on Al speciation and calculation of the Al species generated under the conditions studied. The confocal image results were interpreted by Dr. Bruce Maley, Director of the University of Kentucky Chandler Medical Center Imaging Facility. Conflict of interest: none declared.


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 MATERIALS AND METHODS
 RESULTS
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