Grantee Research Project Results
2003 Progress Report: Intestinal Aluminum Absorption and Bioavailability from Representative Aluminum Species
EPA Grant Number: R829783Title: 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, 2003 through June 30, 2004
Project Amount: $515,720
RFA: Health Effects of Chemical Contaminants in Drinking Water (2001) RFA Text | Recipients Lists
Research Category: Human Health , Drinking Water , 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 on Costar Snapwell polycarbonate filters. A phosphate-free medium at 37°C was used in the chambers on the apical and basolateral sides of the Caco-2 cells. Flux was studied by removing aliquots of medium from both the donor and receiver chambers at multiple time points.
The tightness of the Caco-2 monolayer was measured as transepithelial 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. Al concentration was measured by atomic absorption spectrometry. Lactate dehydrogenase (LDH) release from the Caco-2 cells was measured as an indicator of cytotoxicity and cell lysis. 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 postplating.
Temperature Dependence of Al Uptake Into Caco-2 Cells
To gain insight into the mechanism of Al uptake into Caco-2 cells, we determined the temperature dependence of Al ion, citrate, and fluoride. We calculated the temperature-dependent energy of activation (Ea) from the Arrhenius equation. Ea was used to interpret the uptake process.
Lumogallion Staining and Confocal Microscopic Imaging of Al Localization
To ascertain if the Al associated with the Caco-2 cells in the above studies simply was adsorbed onto the outer surface of the cells or had distributed into the cells, and its intracellular localization, Al localization was conducted using lumogallion, which forms a fluorescent complex with Al. Caco-2 cells were exposed to Al ion, citrate, or fluoride and viewed with confocal and differential interference contrast (DIC) microscopy.
Results
Transcellular flux of each of the five Al species was nearly linear over time, although the rates of flux differed for the five Al species. There is no evidence of saturation of flux. There was a very high correlation between Al and LY flux, suggesting that Al, like LY, crosses Caco-2 cells through the paracellular pathway.
The uptake rate of Al fluoride was greater than the other Al species. The concentration of Al fluoride caused cell toxicity after 90 minutes, as shown by increased LDH release. The uptake rates of Al citrate and maltolate were slower, perhaps caused by inhibition of Al uptake by the ligands.
The Arrhenius plots yielded an Ea for each of the three Al species of 13.3-21.6 kJ/mol (3.2-5.2 kcal/mol), suggesting an active carrier does not mediate Al uptake into Caco-2 cells.
Confocal images of the lumogallion-Al complex showed a similar Al distribution inside the Caco-2 cells after exposure to the three Al species. Al localization is not limited to the outside of the plasma membrane. The lumogallion-Al fluorescence signal was more intense in some intracellular regions. Overlays of the DIC and fluorescent images indicate a high Al concentration associated with cell nuclei.
Future Activities:
We will determine the oral bioavailability of Al ion, citrate, maltolate, and fluoride in the Fisher 344 rat, using 26Al as a tracer for Al. We also will determine the time-course of Al absorption and its associated ligand when Al citrate, maltolate, and fluoride are studied by administration and 14C determination of [14C]-citrate and [14C]-maltolate and analysis of fluoride.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this projectSupplemental Keywords:
accelerator mass spectroscopy, Caco-2 cells, decisionmaking, drinking water, metal absorption, rat, aluminum, Al, neurotoxicity, RFA, Health, Scientific Discipline, Waste, Water, Hydrology, Contaminated Sediments, Environmental Chemistry, Risk Assessments, Environmental Microbiology, Drinking Water, other - exposure, groundwater disinfection, monitoring, ecological risk assessment, metal absorption, aquifer characteristics, human health effects, water quality parameters, exposure and effects, exposure, contaminated sediment, aluminum, chemical contaminants, neurotoxicity, drinking water distribution system, treatment, human exposure, apoptosis, water quality, drinking water contaminants, drinking water treatment, water treatment, aluminum toxicokineticsRelevant Websites:
http://www.mc.uky.edu/pharmacy/faculty/robertyokel.html Exit
http://www.mc.uky.edu/pharmacy/faculty/patrickmcnamara.html Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.