calcein-am and Cadmium-Poisoning

The objective of this study was to investigate whether P-glycoprotein (P-gp) functional expression in intestinal cells is modified after long-term exposure to the food contaminant cadmium (Cd). The Caco-2 cell line, clone TC7, was first validated as a cellular model for long-term exposure to cadmium. Cytotoxicity tests after acute exposure of 24 h showed a significant concentration-dependent decrease in cellular viability at cadmium levels higher than 10 microM and led us to select the cadmium ranges for long-term exposure: 1, 5, and 10 microM. Intestinal cells were exposed to these cadmium concentrations for four consecutive weeks without inducing DNA condensation or fragmentation. In the second part of this work, we studied the functional expression of the drug efflux pump multidrug resistance P-glycoprotein after long-term exposure to cadmium by immunoblotting with the monoclonal antibody F4 and measurement of calcein-AM+/-the P-gp inhibitor verapamil. Western blot analysis with the F4 antibody detected a single band of 170 to 180 kDa which is the size previously reported for P-gp. Calcein-AM assay showed that four weeks exposure of intestinal cells to 1, 5, and 10 microM Cd increased P-gp functional expression in proportion to the Cd concentration.

Topics: ATP Binding Cassette Transporter, Subfamily B; Benzimidazoles; Blotting, Western; Caco-2 Cells; Cadmium Poisoning; Calcium Channel Blockers; Cell Survival; DNA; Drug Resistance, Multiple; Electrophoresis, Agar Gel; Fluoresceins; Fluorescent Dyes; Humans; L-Lactate Dehydrogenase; Neutral Red; Tetrazolium Salts; Thiazoles; Verapamil