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 EC_50s 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.

金属形态对鱼肠金属生物利用度，生物反应性和毒性的作用知之甚少。为了研究这些过程，我们使用了虹鳟（Oncorhynchus mykiss）肠RTgutGC细胞系的体外模型。在组成明确的培养基中，将细胞暴露于两种必需金属（铜和锌）和两种非必需金属（镉和银），从而可以测定溶液中的金属形态。使用基于两个终点的活力测定法测量导致细胞活力降低50％的浓度（EC ₅₀）：代谢活性和膜完整性。在无毒（所有金属为300 nM）和有毒（EC ₁₀）下研究了金属的生物利用度和生物反应性; Ag-0.6，Cu-0.9，Cd-3和Zn-9μM）浓度。通过ICP-MS测定生物利用度（即细胞内金属积累），而通过测量已知的金属暴露（即金属硫蛋白）和铜和铜的生物标志物的mRNA水平，确定生物反应性（即诱导金属特异性转录反应）。锌转运蛋白（即ATP7A和ZnT1）。在曝光介质优势金属种为锌²⁺，CuHP​​O ₄，氯化镉⁺，和氯化银₂ ^-分别为锌，铜，镉，和Ag。EC _50s表现出金属毒性等级：Ag> Cu> Cd> Zn。在RTgutGC细胞中，必需金属的稳态被严格调节，而非必需金属则更容易积累。非必需金属还具有更高的生物反应性，可诱导更高的MT和ZnT1 mRNA水平。综上所述，这些发现表明RTgutGC中的金属毒性不能仅通过细胞外金属形态来解释，而是需要评估金属的生物利用度和生物反应性。