Evaluation of the detoxification mechanisms of metals in aquatic organisms by characterization of hepatic metallothionein (MT)

Abstract

Metallothionein (MT) has been thoroughly used as a biomarker of metals exposure. MT is a low-molecular-weight protein which has many sulfhydryl groups due to the large amount of cysteine in the molecule. These sulfhydryl groups bind a variety of metals and therefore, presumably, make them less toxic to other cellular constituents. However, biochemistry aspects of the protein as isoforms identification and quantification and their specific mechanisms of detoxification in sentinel species are, as yet, weak. Previous data showed that fish, such as the catfish Cathrorops spixil, are efficient bioindicator species to metals contamination in coastal aquatic areas under anthropogenic influence, for instance inputs of Pb, Cd, Hg, Ni, Fe, Zn, Cu and Mn. C. spixil is the most common catfish in the Brazilian coast and has a feeding habit mainly of materials and organisms upon the sediment, where the availability of contaminants is high. In order to understand intrinsic aspects of the detoxification process of toxic metals in sentinel species in natural and anthropogenic conditions, hepatic samples of C. spixii were collected in a non-polluted (Cananeia), as well as one polluted, estuary (Santos-São Vicente) and tested under different assays in order to establish an effective bioanalytical technique to purify the protein like-MT, identify and quantify the specific isoforms and the metal contents in the cell. Therefore, hepatic samples were submitted to ultracentrifugation, thermocoagulation and a chromatographic purification and identification of MT isoforms by size-exclusion-HPLC and anion-exchange-HPLC, respectively. The elution of the protein was made with online UV/Vis detection. Metals in hepatic cytosols were also quantified by HR-ICP-MS. Obtained results showed that the established protocol of bioanalytical technique was effective to purify MT-like protein by SE-HPLC and to identify its isoforms by AE-HPLC. With these analytical strategies, it was possible to identify two kinds of MT isoforms (MT-1 and MT-2) in the C. spixil. Fish MT-1 from polluted areas showed a strong linkage with the levels of Cu, Hg, Pb, Fe and Ni suggesting that the MT-1 is an effective biomarker of metal contamination.