Effect of chloride concentration on the cytotoxicity, bioavailability, and bioreactivity of copper and silver in the rainbow trout gut cell line, RTgutGC.

Abstract

Chloride (Cl-) influences the bioavailability and toxicity of metals in fish, but the mechanisms by which it influences these processes is poorly understood. Here, we investigated the effect of chloride on the cytotoxicity, bioavailability (i.e., accumulation) and bioreactivity (i.e., induction of mRNA levels of metal responsive genes) of copper (Cu) and silver (Ag) in the rainbow trout gut cell line (RTgutGC). Cells were exposed to metals in media with varying Cl- concentrations (0, 1, 5 and 146 mM). Metal speciation in exposure medium was analyzed using Visual MINTEQ software. Cytotoxicity of AgNO3 and CuSO4 was measured based on two endpoints: metabolic activity and membrane integrity. Cells were exposed to 500 nM of AgNO3 and CuSO4 for 24 h in respective media to determine metal bioavailability and bioreactivity. Ag speciation changes from free ionic (Ag+) to neutral (AgCl), to negatively charged chloride complexes (AgCl2-, AgCl3-) with increasing Cl- concentration in exposure media whereas Cu speciation remains in two forms (Cu2+ and CuHPO4) across all media. Chloride does not affect Ag bioavailability but decreases metal toxicity and bioreactivity. Cells exposed to Ag expressed significantly higher metallothionein mRNA levels in low Cl- media (0, 1, and 5 mM) than in high Cl- medium (146 mM). This suggests that chloride complexation reduces silver bioreactivity and toxicity. Conversely, Cu bioavailability and toxicity were higher in the high chloride medium (146 mM) than in the low Cl- (0, 1, and 5 mM) media, supporting the hypothesis that Cu uptake may occur via a chloride dependent mechanism. CLINICAL TRIALS REGISTRATION: This study did not require clinical trial registration.