The role of glutathione and sulfhydryl groups in cadmium uptake by cultures of the rainbow trout RTG-2 cell line

Abstract

The aim of this study is to investigate the role of cellular sulfhydryl and glutathione (GSH) status in cellular cadmium (Cd) accumulation using cultures of the rainbow trout cell line RTG-2. In a first set of experiments, the time course of Cd accumulation in RTG-2 cells exposed to a non-cytotoxic CdCl2 concentration (25 μM) was determined, as were the associated changes in the cellular sulfhydryl status. The cellular levels of total GSH, oxidized glutathione (GSSG), and cysteine were determined with fluorometric high-performance liquid chromatography (HPLC), and the intracellular Cd concentrations were determined with inductively coupled plasma mass spectrometry (ICP-MS). The Cd uptake during the first 24 h of exposure was linear before it approached a plateau at 48 h. The metal accumulation did not cause an alteration in cellular GSH, GSSG, or cysteine levels. In a second set of experiments, we examined whether the cellular sulfhydryl status modulates Cd accumulation. To this end, the following approaches were used: (a) untreated RTG-2 cells as controls, and (b) RTG-2 cells that were either depleted of GSH through pre-exposure to 1 mM L-buthionine-SR-sulfoximine (BSO), an inhibitor of glutathione synthesis, or the cellular sulfhydryl groups were blocked through treatment with 2.5 μM N-ethylmaleimide (NEM). Compared to the control cells, the cells depleted of intracellular GSH showed a 25% reduction in Cd accumulation. Likewise, the Cd accumulation was reduced by 25% in the RTG-2 cells with blocked sulfhydryl groups. However, the 25% decrease in cellular Cd accumulation in the sulfhydryl-manipulated cells was statistically not significantly different from the Cd accumulation in the control cells. The findings of this study suggest that the intracellular sulfhydryl and GSH status, in contrast to their importance for Cd toxicodynamics, is of limited importance for the toxicokinetics of Cd in fish cells.