Size-exclusion HPLC analysis of trace element distributions in hepatic and gill cytosol of Vardar chub (Squalius vardarensis Karaman) from mining impacted rivers in north-eastern Macedonia

Highlights: • Metallomics approach in monitoring effects of metal pollution on Vardar chub • Use of SEC-HPLC/HR ICP-MS for determination of metal distributions within cytosol • Distribution profiles of Cd, Co, Cu, Fe, Mn, Mo, Se and Zn in chub liver and gills • Changes in the distribution profiles due to increased metal exposure level in water • Comparison of metal profiles in Vardar and European chub, two related fish species Many bioindicators have not yet been well characterized regarding their tendency to bind trace elements by different cytosolic biomolecules in response to trace element exposure. Accordingly, our principal aim was to define the cytosolic distributions of Cd, Co, Cu, Fe, Mn, Mo, Se, and Zn among the biomolecules of different molecular masses in liver and gills of Vardar chub (Squalius vardarensis Karaman), a representative fish species of Macedonian rivers, and to determine distribution changes which occur as a consequence of increased exposure to specific trace elements. Additionally, we aimed to confirm the presence of heat-stable biomolecules in chub hepatic and gill cytosols. Distribution profiles were obtained by separation of cytosols and heat-treated cytosols using size-exclusion high performance-liquid chromatography, and by offline determination of trace element concentrations using high resolution inductively coupled plasma-mass spectrometry. Distribution profiles of trace elements were mainly characterized by several peaks encompassing different ranges of molecular masses, as a sign of incorporation of trace elements in various biomolecules within hepatic and gill cytosols. Especially interesting finding was probable binding of Fe to ferritin, which was especially pronounced in the liver, as a sign of important liver function in Fe storage. Furthermore, association with heat-stable proteins, metallothioneins (MT), was indicated for Cd, Cu, and Zn in the hepatic cytosol, as well as for Cd in the gill cytosol, whereas a sign of Zn-MT association was not observed in the gills. The presence of Mo- and Se-binding heat-stable compounds of very low molecular masses (< 10 kDa) in the cytosol was determined for both liver and the gills. Trace elements under all studied conditions were found associated to the same biomolecules, and only their proportions associated to specific cytosolic compounds have changed as a consequence of their increased bioaccumulation in the liver and gills of Vardar chub.