Grantee Research Project Results

2002 Progress Report: Intestinal Aluminum Absorption and Bioavailability from Representative Aluminum Species

EPA Grant Number: R829783
Title: Intestinal Aluminum Absorption and Bioavailability from Representative Aluminum Species
Investigators: Yokel, Robert A. , McNamara, Patrick J.
Institution: University of Kentucky
EPA Project Officer: Page, Angela
Project Period: July 1, 2002 through June 30, 2005 (Extended to June 30, 2006)
Project Period Covered by this Report: July 1, 2002 through June 30, 2003
Project Amount: $515,720
RFA: Health Effects of Chemical Contaminants in Drinking Water (2001) RFA Text |  Recipients Lists
Research Category: Drinking Water , Human Health , Water

Objective:

The objective of this research project is to determine the conditions and routes of intestinal absorption of aluminum (Al) when introduced as a number of different chemical species.

Progress Summary:

Studies of Al Flux Through Caco-2 Cells, Grown on Snapwell Membranes, Using the Vertical Diffusion Chamber

Caco-2 cells were seeded, at passage 20-37, on Costar Snapwell polycarbonate that was 12- mm diameter, 0.4-µm pore size filters. Flux studies were conducted with cells 20-30 days post-plating. A phosphate-free medium at 37°C was used in the chambers and exposed to both the apical (A) and basolateral (B) Caco-2 cell surfaces. Flux in the A-to-B and/or B-to-A direction was studied by removing aliquots from both the donor and receiver chambers at multiple time points.

The tightness of the Caco-2 monolayer was determined by measuring trans-epithelial electrical resistance (TEER) across the Caco-2 cell layer. TEER at the beginning of these studies was approximately 350 ohms/cm² and usually decreased during the flux studies. Aluminum concentration in media and cell lysates was measured by atomic absorption spectrometry. Cell protein was measured by the bioinchoninic acid method. Lactate dehydrogenase (LDH) release from the Caco-2 cells was measured as an indicator of cytotoxicity and cell lysis. To determine the rate of trans-cellular diffusion through Caco-2 cells, DL-[4-³H]propranolol was added to the medium on the apical side of the cells. To determine the rate of paracellular flux between Caco-2 cells, lucifer yellow (LY) was added to the medium on the donor side of the cells.

Studies of Al Uptake Into Caco-2 Cells

Caco-2 cells were plated into six well plates containing 35-mm dishes. Uptake studies were conducted with cells 5-10 days post-plating.

Preliminary Results

• To assess the expression of a marker carrier, P-glycoprotein (P-gp), by the Caco-2 cells, their ability to take up and transport a P-gp substrate, rhodamine 123, introduced on the basolateral side of the Caco-2 cells, was determined 20 to 30 days after plating in the absence and presence of the P-gp inhibitor verapamil. Greater uptake and reduced flux through the Caco-2 cells was seen in the presence of the P-gp inhibitor. These results are consistent with reduced P-gp mediated efflux in the presence of the P-gp inhibitor. The results show expression of P-gp by these cells.

• To address our concern that the citrate introduced as Al citrate might complex Ca (leaving free Al), studies were conducted in the absence of Ca in the uptake media. In the absence of Al citrate, TEER greatly decreased compared to when it was present, and the percentage of LY that moved to the recipient side greatly increased. Therefore, Ca was included in the media in all subsequent studies.

• To characterize the time courses of Al uptake, A-to-B flux studies were conducted for up to 120 minutes in the absence and presence of Al citrate, Al maltolate (Al(ma)3), Al hydroxide, and the Al ion. The Al concentration in the absence of added Al, which averaged 7 ng/ml and was considered to be contamination, was subtracted from the values obtained in the presence of added Al. Flux was linear up to 120 minutes and was greater for Al(ma)3 than Al citrate, both of which were greater than Al ion and Al hydroxide.

• Propranolol and LY flux were determined concurrently. They were found to not correlate. They would not be expected to correlate because of their different routes of flux across Caco-2 cells. Similarly, concurrent determination of propranolol and Al citrate showed that the fluxes did not correlate, suggesting Al citrate did not flux across Caco-2 cells by the same pathway as propranolol. In contrast, concurrent determination of Al citrate and LY flux showed a high correlation, suggesting a similar pathway of flux across Caco-2 cells. These results suggest Al citrate fluxes across Caco-2 cells through the paracellular pathway. Similar high correlations were seen when the flux of the Al ion, Al(ma)3, and Al hydroxide were compared to LY. The permeability of Al hydroxide was lower than the other three Al species when compared to LY. This may be attributed to the limited solubility of Al hydroxide or the presence of charged Al hydroxide species, resulting in a lower concentration of soluble uncharged Al species available to diffuse through the paracellular pathway.

• Flux across the Caco-2 cells in the A-to-B direction was compared to the B-to-A direction. B-to-A flux was greater than A-to-B flux. This observation would not be expected if Al citrate is diffusing at equal rates in each direction across the Caco-2 cells.

• To study the influence of Al, introduced as Al citrate, on the integrity of Caco-2 cell tight junctions, the effect of various Al citrate concentrations, introduced on either the A or B side, on LY permeation was studied. Increasing concentrations of Al citrate increased the apparent permeability of LY. Increased paracellular permeability was greater when Al was introduced on the B side. These results suggest that the greater B-to-A Al permeability might be due to Al-induced disruption of the paracellular pathway, permitting greater Al permeation through that pathway.

• To determine the time course of Al uptake in Caco-2 cells, studies were conducted with Al citrate and Al(ma)3, followed by washes of the cells that contained either no or 2 mM Ethylenediamine Tetraacetic Acid (EDTA). EDTA was included to remove Al associated with labile storage sites, such as that adsorbed to the cell surface and possibly intracellular sites. The time course of Al maltolate uptake was linear, irrespective of the absence or presence of EDTA in the washout medium. Inclusion of EDTA, however, resulted in less Al associated with the Caco-2 cells after Al citrate exposure, suggesting Al uptake into at least two compartments from Al citrate, including one that is labile.

• Uptake experiments were conducted with 4-hours exposure to Al citrate and Al(ma)3. The cell/media Al ratio was 0.7 and 1.9, respectively, suggesting concentrative uptake of Al(ma)3. Uptake after 7 hours exposure to Al citrate was nonlinear, but was linear after Al(ma)3 exposure, producing cell/medium ratios of 1.0 and 4.0, respectively. Exposure of Caco-2 cells to Al citrate for 12 and 18 hours resulted in much more cell detachment from the dish than seen with Al(ma)3 exposure, suggesting greater toxicity.

• Comparison of the uptake of Al citrate and Al(ma)3 to their A-to-B flux after 2 hours, suggested uptake was approximately 25 percent and 40 percent of flux, based on comparable cell surface areas. This comparison indicates that Al primarily fluxes across Caco-2 cells through the paracellular pathway, and not trans-cellularly.

Future Activities:

Future activities are to continue and complete the flux and uptake studies. Specifically, we will: (1) conduct further replicates of the ongoing studies of Al ion and Al hydroxide A-to-B flux; (2) conduct studies of Al fluoride flux in the presence of LY to ascertain if each of these Al species permeate Caco-2 cells primarily by diffusion; (3) conduct further studies of Al citrate and Al(ma)3 uptake at exposure times greater than 4 hours to ascertain the saturability of Al uptake and the concentrative uptake ratio; (4) further pursue the preliminary results suggesting active transport of Al(ma)3 into Caco-2 cells; and (5) begin in vivo studies to determine the oral bioavailability of some, perhaps all, of the five Al species, as proposed in the grant application for this project.

Journal Articles:

No journal articles submitted with this report: View all 8 publications for this project

Supplemental Keywords:

accelerator mass spectroscopy, Caco-2 cells, decisionmaking, drinking water, metal absorption, rat, aluminum, Al, metal absorption, neurotoxicity., RFA, Health, Scientific Discipline, Water, Waste, Risk Assessments, Environmental Chemistry, Contaminated Sediments, Environmental Microbiology, Hydrology, Drinking Water, other - exposure, aluminum, aluminum toxicokinetics, treatment, contaminated sediment, ecological risk assessment, water quality, water quality parameters, human exposure, metal absorption, chemical contaminants, groundwater disinfection, neurotoxicity, monitoring, exposure, drinking water contaminants, drinking water treatment, apoptosis, water treatment, human health effects, human health risk, aquifer characteristics, drinking water distribution system

Relevant Websites:

http://www.mc.uky.edu/Pharmacy/faculty/yokel/ Exit
http://www.mc.uky.edu/Pharmacy/faculty/mcnamara/ Exit

Progress and Final Reports:

Original Abstract

2003 Progress Report

2004 Progress Report

2005

Final Report