The role of metal speciation on metal bioavailability, bio-reactivity and toxicity at the fish intestine is poorly understood. To investigate these processes, we used an in vitro model of the rainbow trout (Oncorhynchus mykiss) intestine, the RTgutGC cell line. Cells were exposed to two essential metals (copper and zinc) and two non-essential metals (cadmium and silver) in a medium of well-defined composition, which allowed the determination of metal speciation in solution. Concentrations resulting in a 50% cell viability reduction (EC50) were measured using a viability assay based on two endpoints: metabolic activity and membrane integrity. Metal bioavailability and bio-reactivity was studied at non-toxic (300 nM all metals) and toxic (EC10; Ag-0.6, Cu-0.9, Cd-3, and Zn-9 μM) concentrations. Bioavailability (i.e. intracellular metal accumulation) was determined by ICP-MS, while bio-reactivity (i.e. induction of a metal specific transcriptional response) was determined by measuring the mRNA levels of a known biomarker of metal exposure (i.e. metallothionein) and of copper and zinc transporters (i.e. ATP7A and ZnT1). Dominant metal species in the exposure medium were Zn2+, CuHPO4, CdCl+, and AgCl2- respectively for Zn, Cu, Cd, and Ag. The EC50s showed the metal toxicity hierarchy: Ag > Cu > Cd > Zn. In RTgutGC cells, essential metal homeostasis was tightly regulated while non-essential metals accumulated more readily. Non-essential metals were also more bio-reactive inducing higher MT and ZnT1 mRNA levels. Taken together these findings indicate that metal toxicity in RTgutGC cannot solely be explained by extracellular metal speciation but requires the evaluation of metal bioavailability and bio-reactivity.
Keywords: In vitro alternative; Metal bioavailability; Metal bioreactivity; Metal speciation; Metal toxicity.
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