Effects of Glutathione Depletion on the Cytotoxic Actions of Cadmium in LLC-PK 1 Cells

Abstract

Recent studies have shown that exposure of LLC-PK 1 cells to micromolar concentrations of Cd 2+ for 1-4 hr causes the disruption of the junctions between the cells, whereas exposure to higher concentrations of Cd 2+ for longer periods of time causes more severe toxic effects and cell death. Studies suggesting that glutathione may serve a protective role against Cd 2+ toxicity in other tissues and cells led us to examine the effects of glutathione depletion on the cytotoxic actions of Cd 2+ in the LLC-PK 1 cell line. Confluent cells on Falcon cell culture inserts were depleted of glutathione by exposing them to 250 μM buthionine sulfoximine for 18 hr and then exposed to various concentrations of Cd 2+ for up to 24 hr. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the trans-epithelial electrical resistance. Cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. The results showed that depleting the cells of glutathione did not alter the early junction-perturbing effects of Cd 2+, but greatly enhanced the lethal effects. In both the glutathione-depleted and the normal cells, junctional changes were evident after as little as 1 hr of Cd 2+ exposure. While the normal cells did not begin to die until they had been exposed to Cd 2+ for 12-24 hr, the glutathione-depleted cells began to die after only 8 hr of Cd 2+ exposure. Additional results showed that Cd 2+ exposure had no effect on the total levels of glutathione at the time in which the junctional effects were occurring, but caused a marked decrease in glutathione levels at the time the cells were dying. These results indicate that the early junctional effects of Cd 2+ do not result from alterations in intracellular glutathione or sulfhydryl metabolism, whereas the more severe cytotoxic effects and cell death may involve glutathione-sensitive mechanisms.