engleski

Dribution of Cu, Fe and Cd among SE-HPLC separated cytosolic proteins isolated from liver, gills and intestinal parasite of European chub (Squalius cephalus L.)

Metal contamination in freshwater ecosystem is a critical environmental issue due to metal uptake, accumulation and possible toxicity in many aquatic organisms. Fish are one of the most indicative species in freshwaters, for the estimation of trace metal pollution and possible risk to human health. The pattern of metal distribution in fish depends on ambient metal concentration, its speciation and the uptake routes, which involve dietborne (intestine) and waterborne (gills) metal exposure, followed by blood transfer to target organs for metal storage and detoxification (liver and kidney). Trace metal detoxification processes depend mainly on metal binding to metallothioneins, inducible cellular proteins which reduce toxic metal effects (Hg, Cd, Ag) and take part in homeostasis of essential metals (Zn, Cu). To elucidate the metabolism, bioavailability and toxicity of metals in the aquatic environment, it is not sufficient to determine total tissue or cytosolic metal concentrations, since metals in living organisms form part of bigger, more complicated molecules, mainly proteins. So far, biological functions and mechanisms of metal toxicity in different organisms are still not thoroughly investigated, and the proteins to which they bind are only partially identified. In addition to already established assessment of metal exposure, by analysing metal and metallothionein concentrations in fish indicator organs, investigation of metal subcellular partitioning, involving proteins other than metallothioneins, could provide a more complete understanding of potential mechanisms of metal toxicity. Moreover, during the last few decades it is discovered that fish intestinal parasites (class Acanthocephala) effectively accumulate metals, especially toxic ones. Although metal concentrations in parasites highly exceed those in fish tissues, there is no information on metal distribution or detoxification in acanthocephalans. The aim of this study was to compare the distribution of essential (Cu, Fe) and nonessential metals (Cd) among cytosolic protein fractions of gills (metal uptake site), liver (metal detoxification site) and acanthocephalans (Pomphorhynchus laevis) of European chub from the Sutla River, Croatia. The fractionation of cytosols was carried out by size exclusion high performance liquid chromatography (SE-HPLC ; Superdex™ 200 10/300 GL column), and metal measurements in collected chromatographic fractions were performed off-line by high resolution inductively coupled plasma mass spectrometry (HR ICP-MS). In conditions of low metal contaminated river water, basic distributions of analysed metals indicated metal association with specific proteins, for Cu and Cd with metal binding protein, metallothionein and for Fe with Fe-storage protein, ferritin. The obtained information within this study presents the starting point for identification and characterization of specific metal-binding proteins in chub and acanthocephalans, which could be further on used as markers of metal exposure or effect on fish.

chub ; acanthocephalans ; metal distribution ; cytosolic proteins ; SE-HPLC

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