Видоспецифічне різноманіття участі металотіонеїнів прісноводних тварин у регуляції гомеостазу металів

УДК 597:554.3.13


O. B. Stolyar, H. I. Falfushynska, V. V. Nykyforov

Hnatyuk Ternopil National Pedagogical University, Ternopil, Ukraine, E-mail: stolyar@tnpu.edu.ua

Key words: metallothioneins, freshwater animals, copper, zinc

О. Б. Столяр, Г. І. Фальфушинська, В. В. Никифоров

Тернопільський національний педагогічний університет ім. В. Гнатюка, м. Тернопіль, Україна,
E-mail: stolyar@tnpu.edu.ua

Ключові слова: металотіонеїни, прісноводні тварини, мідь, цинк

Metallothioneins (MTs) are the thermostable sulphydryl–rich proteins. Its biological role is associated with the homeostasis of essential metals zinc in vertebrate, copper in some unicellular organism and nonessential metal cadmium in wide range of organisms by its chelating in non-toxic complexes (Kagi, Schaffer, 1988). For the last decade the MTs role in copper binding in some Crustacea and terrestrial gastropod was elucidated (Brouwer et al., 2002, Dallinger et al., 2004). But it is luck of information about comparative effectiveness of the participation of MTs of different animal species in the metals binding under environmental pollution. Particularly it has connected to freshwater animals. Thus it’s respect an interesting to compare the participation of MTs of freshwater animal species with different physiological requirement – carp (Cyprinus carpio Linnaeus, 1758), broad-fingered crayfish (Astacus leptodactylus Eschscholtz, 1823), and bivalve mollusc Anodonta (Anodonta cygnea Linnaeus, 1758) – in the binding of zinc and copper after the pollution of water in ecologically realistic concentrations.

Individuals of the carp, crayfish and mollusc were collected in summer from Ternopil regional fish centre lakes (Zalisci), Ukraine. The first group of each species (control) received no treatment, another groups were exposed to Cu2+, Zn2+, Mn2+, Pb2+ at nominal concentrations 0,01, 0,1, 0,12 and 0,01 mg·l–1 respectively, its mix in the same concentrations or 0,02 mg·l–1 phenol during 14 days in the aerated 200 l tanks. The concentrations of metals used in this study were selected to mimic concentrations as found in the fresh water (Ukraine). After exposure five individuals in each group were dissected and tissues were removed. We were used the liver of carp, hepatopancreas of crayfish and digestive gland of mollusc. MTs were obtained from thermostable material of tissue by consequent gel–permeation and ion–exchange chromatography. The content of zinc and copper was measured in total tissue, thermostable proteins, isoforms of MTs and calculated in thermolabile pellet. The activity of Cu,Zn-superoxide dismutase (SOD; EC was also measured.

The distribution of metals among MTs and another tissues compounds shown the great difference between carp and invertebrate animals (tabl.). Under the ion–exchange chromatography MTs were shown to comprise of two distinct proteins. They are designated as MT–1 and MT–2 based on its chromatographic behaviour. MTs of all species had the similarity of UV–spectra with high level of absorption in the middle UV, indicative the presence of characteristic metals–thiolate clusters. At the same time it was revealed the great difference in the elution value proportions
(VMT–1/VMT–2) among species. So for carp predominate form was MT–2, and for crayfish and mollusc – MT–1. According to metals content carp’s MTs reveale to be Zn–MTs, thus crayfish’s and mollusc’s one – Zn,Cu–MTs. The activity of Cu,Zn–SOD in carp was fife times lesser as in crayfish and mollusc (0,19±0,01, 1,20±0,17 and 1,25±0,17 units/mg proteins respectively).

The differences in answer of MTs and SOD of treated animals has been determined. At first it connects to VMT–1/VMT–2 changes. After the effect of each contaminant on carp the depletion of major MTs form (MT–2) was observed. The profile of MTs of crayfish’s wasn’t changed significantly by any treatment. The action of copper on Anodonta provoked the significant increasing of MT–2 value. Differences in the binding of metals to MTs were observed too. Under the effect of all waterborne contaminants the carp’s MTs successfully maintained the constant content of zinc but the copper content in MT was sharply reduced (to 30–50 % of total level in MTs). Among isoprotens the role of MT–1 in metals binding was comparatively increased, but participation of main fraction – MT–2 was significantly decreased. The MTs of crayfish and Anodonta bound successfully the excess of zinc after all kind of treatment, especially after manganese effect. At the same time, copper content maintained at a rather stable level, or even enhanced at copper and mixed–metal action. The activity of Cu,Zn–SOD is depending on the supplying of copper in suitable form. In carp it was significantly decreased by any kind of treatment, whilst in crayfish and mollusc it was stable or elevated. The elevation of unbound with MTs copper level in tissue was observed in fish, especially after the lead and phenol effect.

Table. The content of copper and zinc in the metallothioneins of freshwater animals,
nmol·g–1 of tissue (% of total metal in tissue)

MTs isoforms











12 (12)

130 (6)

34 (8) 77 (7) 17 (21) 46 (15)


41 (40)

172 (8) 33 (8) 69 (6) 24 (29) 39 (13)


Thus, our studies revealed significant differences in copper and zinc homeostasis among three investigated species. It seems to be a function of their evolutionary state more than effected metal’s kind. The greatest differences among species’ MTs were observed in its copper binding capacity. The suppression of copper–binding function in the main MTs isoprotein in carp, its stability in crayfish’s MTs and enhanced capacity in Anodonta’s MTs after different treatments may be explained on the specificity of copper metabolic function in these species. The difference in the Cu,Zn–SOD activity and sensitivity among species confirms this conclusion. As known, copper is the one of most toxic metal for fishes. In Crustacea MTs participate in copper supplying for haemocyanin function. At last Anodonta is the mollusc species in which respiratory pigment in haemolymph is absent in general (Kreps, Smirnov, 1937). But bivalve mollusc is a sessile filter, which can effectively concentrate pollutants. The most stability of zinc–binding capacity was revealed in fish, which had the perfect regulation of it homeostasis in liver with MTs participation (Olsson et al., 1989). Invertebrate animals demonstrated the remarkable effectiveness in the binding of zinc excess. The common level of copper and zinc in tissues or its level in pellet and sertain thermostable compounds wasn’t changed consequently and significantly after the treatment of animals.

Thus, our findings allow consideration of great differences in copper and zinc homeostasis in animals’ with participation of MTs and Cu,Zn–SOD. It seems to be a function of their evolutionary state more than metal’s kind. Thus the copper sequestration with MTs participation in carp tissues may provide a sensitive indication of stress induced by the presence of toxicants. So the depletion of copper relative function of MTs and Cu,Zn–SOD was the most general and consecutive responses to environmental stress in fish. The activation of zinc and copper binding function of MTs was the remarkable feature of stress in crayfish and bivalve mollusk. The species–specifically behaviour of MTs and SOD of freshwater animals’ behaviour must be taking into account in the biomonitoring of water quality.

This work was supported by State budget Projects 0101UO00303 from the Ministry of Education and Science of Ukraine.

Zoocenosis — 2005
 Біорізноманіття та роль зооценозу в природних і антропогенних екосистемах: Матеріали ІІІ Міжнародної наукової конференції. – Д.: Вид-во ДНУ, 2005. – С. 55-57.