Reactivity of metallothioneins of frog Rana ridibunda treated by copper and zinc ions.

The metal-buffering and stress proteins metallothioneins (MTs) of frog are characterised by unusually high content of copper as for vertebrate animals and instability that was shown in our previous studies. They easily lost copper and especially zinc under unfavourable conditions. The aim of this study was to examine the reactivity of SH groups in the MTs from the liver of frog Rana ridibunda after the effect of Cu2+ (0.01 mg/l) and Zn2+ (0.1 mg/l) ions on the organism during 14 days. The alpha- and beta-domains of MTs with molecular weights of about 4 kDa were separated by the size-exclusion chromatography on Sephadex G-50. Unlike higher vertebrates, frogs demonstrated higher reactivity of alpha-domain than beta-domain with the Ellman's reagent (DTNB). The signs of partial oxidations in beta-domain included the creation of by-products with molecular weight about 12 kDa, low reactivity of SH-groups, and typical of -S-S-bonds peculiarities of UV-spectra. The effect of both metal ions on frog provoked the elevation of SH-groups reactivity in a-domain with the appearance of by-product with molecular weight of 16 kDa and its reduction in beta-domain. The incubation of MTs of control animals with 0.5 and 5.0 mM of H2O2 did not affect its chromatographic characteristics. In the frogs loaded by Cu2+ and Zn2+ the effect of 5.0 mM H2O2 on MTs provoked the release of 4 kDa product. So the alpha-domain is responsible for the increased release of metals from injured MTs in frogs, whereas extremely high oxidizability of beta-domain makes its participation in the exchange of metals elusive and provokes the aggregation of MTs.

Comparison of metal bioavailability in frogs from urban and rural sites of Western Ukraine.

The seasonal and spatial fluctuations of heavy metals in the liver of the frog Rana ridibunda from a river in Western Ukraine were investigated. Liver weight was seen to increase from spring to summer/autumn, most probably as a result of accumulation of metabolites, especially fat and glycogen. The concentrations of the metals found in the liver of the frog was in the order: Fe>Cu approximately Zn>Mn>Cd. For most metals, the highest concentration was recorded in the frogs inhabiting the urban site. The highest level of Cu in the liver was observed in the spring, in the agricultural site, while the highest level of other metals was observed in the summer. The most probable explanation for the high concentration of Cu in the rural site was that in this wetland there were discharge effluents from fungicides with Cu in their formula. Compared to other metals, the bioavailability of Cu was approximately 1000 times higher. The high concentration of Fe rather reflects its fluctuation in the water. Despite its very low concentration in the water (below the limit of detection), Cd was detected in the liver of frogs inhabiting both sites. This is an indication that tissues accumulate Cd, despite the very low concentration detected in the water. This may be an indication of intermittent exposure of frogs to Cd and possibly of other heavy metals.