Induction and binding of Cd, Cu, and Zn to metallothionein in carp (Cyprinus carpio) using HPLC-ICP-TOFMS

The binding of Cd, Cu, and Zn to metallothionein in carp was studied under control and acute Cd exposure scenarios. Carp were exposed to different Cd concentrations for 96 h. Total (Cu, Cd, Zn)-MT levels were determined by the cadmium thiomolybdate saturation assay. Total tissue and cytosolic Cd, Cu, and Zn concentrations were determined by ICP-MS. The cytosolic metal speciation was determined by high pressure liquid chromatography (size-exclusion [SE] in combination with anion exchange [AE]) directly coupled to an inductively coupled plasma time of flight mass spectrometer (ICP-TOFMS). This coupled technique allows the chromatographic separation and online determination of the metals associated to the protein fractions separated. Very strong differences in the tissue compartmentalization and cytosolic speciation of the metals were observed. For example, over 30% of cytosolic zinc was bound to MT in liver while this was only 2% in the kidneys although total cytosolic levels were considerably higher. Induction of metallothionein during cadmium exposure was also tissue specific, displaying different response patterns in gills, liver, and kidney. Cadmium accumulated much stronger in liver and kidney compared to the gills and the latter also showed much lower MT levels. The renal MT-induction was more sensitive to Cd exposure than the hepatic MT induction since a significant increase of Cd-MT and total MT levels occurred at lower tissue Cd concentrations in the kidney in comparison to the liver, except for the highest Cd exposure level where a drastic 10-fold increase in hepatic Cd-MT was observed. At this Cd exposure level also an apparent spill over of zinc to the high molecular weight fraction was observed in the kidneys.