Shapes of Differential Pulse Voltammograms and Level of Metallothionein at Different Animal Species.

Metallothioneins play a key role in maintaining homeostasis of essential metals and in protecting of cells against metal toxicity as well as oxidative damaging. Excepting humans, blood levels of metallothionein have not yet been reported from any animal species. Blood plasma samples of 9 animal species were analysed by the adsorptive transfer stripping technique to obtain species specific voltammograms. Quite distinct records were obtained from the Takin (Budorcas taxicolor), while other interesting records were observed in samples from the European Bison (Bison bonasus bonasus) and the Red-eared Slider (Trachemys scripta elegans). To quantify metallothionein the catalytic peak Cat2 was used, well developed in the Domestic Fowl (Gallus gallus f. domestica) and showing a very low signal in the Red Deer (Cervus elaphus). The highest levels of metallothionein reaching over 20 µM were found in the Domestic Fowl. High levels of MT were also found in the Bearded Dragon (Pogona vitticeps) and the Grey Wolf (Canis lupus lupus). The lowest values of about 1-3 µM were determined in the Red-eared Slider, Takin and Red Deer. Employing a simple electrochemical detection it was possible to examine variation in blood metallothionein in different species of vertebrates.


Introduction
Regular cellular functions depend on the stability and minimal changes of the inner environment. Therefore it is not surprising that there are numerous regulatory mechanisms maintaining the inner environment in higher animals. The most extensively studied homeostatic mechanism is probably the buffering ability of body fluids maintaining optimum concentration of acids and alkalis (i.e. stability of pH). All these homeostatic mechanisms are strictly controlled and many of them employ the negative feedback. It may be supposed that levels of heavy metal ions are also strictly controlled in all living organisms [1,2]. Free ions can cause many serious problems including oxidative stress or permanent signalling within the cell [3]. The bond between metals and albumin, though it is thought to be nonspecific, is important for the maintenance of homeostasis of metal ions. Specific proteins binding metal ions such as iron (ferritin) or copper (ceruloplasmin) have also been described.
One intracellular heavy metal binding protein, metallothionein (MT), was discovered in vertebrates in 1957 [4]. Its molecular weight ranges from 6 to 7 kDa in all mammalian species and it may be characterised by a unique content of metal ions as well as sulphur [5]. It has a high affinity for various metal ions which may represent up to 20% of the metallothionein weight. Phylogenetic relations of various MT families were obtained by protein and gene sequencing. Based on the results obtained phylogenetically related families and subfamilies of evolutionary similar complexes of MT families in vertebrates can be distinguished ( Figure 1). Primarily metallothioneins were divided into three classes. Class I includes MTs found in various mammalian species. Class II comprises all other protein MTs not belonging into class I. Class III contains metalloisopolypeptids with a gamma-glutamyl-cysteine unit. Sequentional similarity, however, may not be sufficient for the classification of MTs. Therefore, a definition of MT clans has been proposed. Clans unite MTs according to other characteristics such as their general structure, thermodynamics and metals-binding groups.
Metallothioneins are present throughout the animal kingdom and have also been found in higher plants, eukaryotic micro-organisms and many prokaryotes [5][6][7]. In animals they occur in tissues such as liver, kidney, pancreas and intestines, in particular [8][9][10][11]. There are considerable differences in the content of MT related to species, tissues, age, nutrition and other not well known factors [12][13][14][15][16][17]. Moreover, heavy metal ions in the environment play an important role. Their effects, however, are not fully understood [18][19][20][21]. There are probably constant levels of metallothionein in a non-stressed cell. Interestingly, metallothionein may accumulate within lysosomes and has also been found in the cellular nucleus [22][23][24][25]. Although metallothionein has been identified in a broad range of species, to our knowledge there has been published only one paper comparing systematic differences in various mammals including humans concerning species variation in hepatic metallothionein [26]. In the aforementioned study, however, tissue samples for metallothionein analysis were obtained from dead necropsied specimens. As blood levels of metallothionein might be of clinical importance, the objective of this study was to determine blood plasma levels of metallothionein of several species of vertebrates using electrochemical detection (adsorptive transfer stripping technique with differential pulse voltammetry Brdicka reaction -AdTS DPV Brdicka reaction), but the samples were collected from live specimens prior to analysis. The species studied include Red-eared Slider, Bearded Dragon, Domestic Fowl, Grey Wolf, Bactrian Camel, Reindeer, Takin, European Bison, and Red Deer.

Chemicals
Rabbit liver MT (MW 7143), containing 5.9 % Cd and 0.5 % Zn, was purchased from Sigma Aldrich (St. Louis, USA). Tris(2-carboxyethyl)phosphine (TCEP) was produced by Molecular Probes (Evgen, Oregon, USA). All other chemicals used were purchased from Sigma Aldrich unless noted otherwise. The stock standard solutions of MT at 10 μg/ml with 1 mM TCEP [27][28][29] were prepared with ACS water (Sigma-Aldrich, USA) and stored in the dark at -20 °C. Working standard solutions were prepared daily by dilution of the stock solutions.

Electrochemical measurements
Electrochemical measurements were performed with AUTOLAB Analyser (EcoChemie, Netherlands) connected to VA-Stand 663 (Metrohm, Switzerland), using a standard cell with three electrodes. A hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm 2 was used as working electrode. An Ag/AgCl/3M KCl served as reference electrode and glassy carbon electrode as auxiliary electrode. For smoothing and baseline correction the software GPES 4.4 supplied by EcoChemie was employed.  250 mV, E ads = 0 V. Temperature of supporting electrolyte was 4 °C (Julabo, Germany). For other details see [31,32,40,41].

Species examined
Nine different species of vertebrates were used for the comparative study. These included two reptilian species (Reptilia), i.e. Red-eared Slider (Trachemys scripta elegans) and Bearded Dragon

Samples and their preparation prior metallothionein analysis
Blood plasma samples of several zoo animals (ZOO Brno, Czech Republic) and game birds (Game Bird Farm Jinacovice, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic) were used for analyses. Blood was collected during routine health status check from vena ulnaris (birds) or vena jugularis using a hypodermic needle and a syringe in the volume not threatening the animal. Heparin (HEPARIN LÉČIVA 5000 iu/1 ml) was used as an anticoagulant. Immediately after collection, blood was centrifuged for 5 min at 3000 g. Then, plasma was separated and frozen in 1.5ml Eppendorf vials prior further analysis. The sample was prepared by heat treatment. Briefly, the sample was kept at 99 °C in a thermomixer (Eppendorf 5430, USA) for 15 min with occasional stirring, and then cooled to 4 o C. The denatured homogenates were centrifuged at 4 o C, 15 000 g for 30 min. (Eppendorf 5402, USA). Heat treatment effectively denatures and removes high molecular weight proteins out from samples [35,42].

Statistical analysis
MICROSOFT EXCEL® (USA) was used for statistical analyses. Results are expressed as mean ± S.D. unless noted otherwise. Differences with p < 0.05 were considered significant.

Shape of DP voltammetric curves of mammalian species analysed
Prepared blood plasma samples of individual animal species were analysed by the adsorptive transfer stripping technique in 5 µl drop of the sample (Figure 2). In this way, species specific voltammograms were measured. Typical DP voltammograms of samples from the animals species analysed are shown in Figure 3. Quite distinct records were obtained from the Takin (Budorcas taxicolor), while other interesting records were observed in samples from the European Bison (Bison bonasus bonasus) and the Red-eared Slider (Trachemys scripta elegans). Moreover the shapes of DP voltammetric curves are so called specie-characteristic. Based on the shape of the curve the animal specie can be identified (patent PV 2007-568).
The Brdicka reaction can be considered as a suitable technique to determine proteins containing the thiol group. Catalytic signals of hydrogen separation provided by proteins on the mercury electrode in the presence of ammonium buffer (1 M NH 4 Cl + NH 4 OH) with content of the cobalt salt Co(NH 3 ) 6 Cl 3 are used [32,[43][44][45]. Metallothionein gives four distinct signals Co1, RS 2 Co, Cat1, and Cat2 on the electrochemical record. Recently it has been shown that this technique may be utilized for determination of metallothionein in whole blood and blood sera of humans [32,46]. To quantify metallothionein we used the catalytic peak Cat2, well developed in the samples from Domestic Fowl

Association of changes in metallothionein level in blood of the vertebrates and their taxonomic group
As shown above, this study evaluated differences in blood levels of metallothionein in 9 animal species belonging to rather unrelated taxonomic groups. Comparison of results with references can be a problem because the only study concerning species variation in metallothionein concerns its hepatic levels and a different list of 15 species examined [26]. It is clear that the highest blood MT levels are in birds, insectivorous lizards and carnivores. Lower MT levels are in herbivorous animals, of which the Red Deer and Takin show the lowest MT findings. Henry et al. found the lowest MT levels in all examined rodents [26]. This taxonomic group, however, was not the subject of the present study. Marked species differences may be hypothetically explained by different roles of metallothioneins in the organism such as homeostasis of essential metals including zinc and copper, protection against metal toxicity and oxidative damage [47]. In this respect, organisms of higher trophic levels (birds, lizards, carnivores) may be supposed to be more subjected to the action of environmental and nutritional stressors, needing thus higher levels of MTs for the maintenance of body homeostasis. On the other hand, the results may be expected to really express species differences in blood metallothioneins because samples collected from non-stressed animals only were included to the study. Considering heavy metals, the main way of entrance into the organism is that by ingestion. Plants, as the essential food source of herbivores, may become contaminated from the atmosphere and soils. Lichens and mosses, for example, belong to susceptible bio-indicators of environmental pollution by heavy metals [48][49][50][51]. Moreover, the Reindeer are trophic specialists preferring this feed [52,53].   Free-ranging European Bisons have been found to accumulate more cadmium in the liver and kidneys than domestic cattle [54] and, actually, this species was found with twice higher levels of metallothionein than the Takin belonging to the same family of Bovidae. Apart from lichens and mosses, direct bio-indication of heavy metals is possible using some mushrooms, herbs, trees and shrubs [55]. The above listed items are commonly included in the forage of herbivores. Contrary to terrestrial ecosystems, there are many studies on heavy metals in different aquatic trophic levels. Much attention has been paid to heavy metals in fish; with the highest levels determined in predatory species [56][57][58][59]. Interestingly, the only aquatic species evaluated in this study, i.e. the Red-eared Slider, showed low levels of metallothionein.

Conclusion
Blood levels of metallothionein have not yet been reported from any animal species except humans. This study reports on study of variation in blood level of metallothionein in different species of vertebrates. It can be concluded that metallothionein levels depend on the trophic level of the organism.