UDC: 577.29:564.38:57.014 EVALUATION OF METALLOTHIONEIN RESPONSE IN LYMNAEA STAGNALIS POND SNAIL EXPOSURED TO TRACE METALS AND THIOCARBAMATE FUNGICIDE

© 2019 L. L. Gnatyshyna. Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org/ and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 577.29:564.38:57.014

Pulmonate mollusks posses high uptake rates of dissolved metals and their bonding, mainly Cd, Zn and Cu by the metal-buffering protein, metallothionein (MT). However, most investigations are focused at the terrestrial species, whereas aquatic snails are studied scantly. Therefore, the main goal of our study was to evaluate a specificity of metal-binding function of the metallothionein in Lymnaea stagnalis pond snail. The mollusks were subjected to typical metallothionein inducers Cd 2+ , 15 µg·L -1 ; Cu 2+ , 10 µg·L -1 ; Zn 2+ , 130 µg·L -1 and oxidative stress inducer thiocarbamate Tattoo fungicide, 91 µg L −1 during 14 days. Applied concentrations corresponded to the ecologically relevant concentrations. Metal concentrations and metallothionein characteristics in a digestive gland were analyzed. In present study, all exposures to metals caused the elevation of the concentration of metals (Zn, Cu or Cd) in tissues and the total level of metallothionein detected from their thiols (MT-SH). Particularly, Zn level in tissues was increased by ~2−3 times in all cases of the exposure to metals. Cd increased the level of metal-contained form of the metallothionein (MT-Me). Tattoo affected the levels of metals and MT-SH in the tissue to low extent but caused the redistribution of metals between the metallothionein (increase) and other compounds. The distortion of the elution profile of metallothionein on the DEAE-cellulose was shown at each exposure. To summarize, snails demonstrated a wide spectrum of responses and juxtaposition of different characteristics depending on the exposure. The responses to metals and

INTRODUCTION
Freshwater snails are an important species in the aquatic ecosystems due to their rapid grazing rates, sensitivity to different kinds of pollution [3,5,17,24,28]. At present, more studies address the values and services of freshwater snails within an ecosystem, their ability to accumulate either metals or metal-containing compounds and a risk to higher trophic levels [32]. Freshwater pulmonate mollusk, pond snail Lymnaea stagnalis (Linné, 1758), as known as the "great pond snail," represents the predominate species in the freshwater zoobenthos of Central Europe [15].
Metallothionein (MT) is the most acknowledged аmong the biomarkers of pollution in mollusks, as the indicator of specific pollution by highly toxic metal cadmium due to high affinity of this metal-binding protein to cadmium [1,35]. In the polluted environment, this metal of the anthropogenic origin can replace physiological metals zinc (Zn) and copper (Cu) in metal-thiolate clusters of the MT. Moreover, it is one of the most effective inducers of MT expression in different aquatic organisms [1]. It is noteworthy to mention that MT of the terrestrial snails are investigated thoroughly, particularly by R. Dallinger and colleagues [5,18,24,27]. They demonstrated especially high variability of the molecular forms induced by the exposures to cadmium and copper. However, the responses of metallothionein in snails to other substances as the essential targets for these proteins are unknown. Among the aquatic mollusks, the MT in bivalve mollusks were shown to enhance the total concentration without an increase in the level of metalcontained form in different exposures, including thiocarbamate and cobalt-contained nanonized polymer [10,11]. Indeed, about 30 % of amino acid composition of metallothionein is represented by cysteine. Therefore, a vulnerability of metal binding function despite high total level of protein is expected from the particular metal binding/release dynamics intrinsic to these proteins [35].
Due to a lack of information concerning the properties of the MT in the aquatic snails, the goal of this study was to evaluate the peculiarities of the metal-binding response of the MT in the freshwater pulmonate L. stagnalis mollusk pond snail. The snails were subjected to typical metallothionein inducers, namely Cd, Zn, Cu, and known oxidative stress inducer and metal chelator thiocarbamate fungicide [4,7,33].

MATERIALS AND METHODS
The specimen collection was carried out at turn of August, when the reproductive system is not significantly affected. About 50 adult specimens of the Lymnaea stagnalis pond snail with shell height of 25-35 mm were collected in the pristine area located in the upstream portion of river Seret (Ternopil region, Ukraine, 49°49′ N, 25°23′ E). Individuals were transported to the laboratory in cages with native water and adapted to aerated, softened tap water with continued support of mineral/salt balance (17 ± 1 °C, pH 7.3 ± 0.2, CaCO 3 level 86.8 ± 1.0 mg·L -1 , dissolved oxygen level 8.67 ± 0.51 mg·L -1 , ammonia (NH 3 /NH 4 + ) and nitrite levels below 0.1 mg·L -1 ) for seven days. After that, they were divided in five groups, control group (C), and groups subjected to waterborne metals copper (Cu 2+ , 10 µg·L -1 correspondent to 0.156 µM), zinc (Zn 2+ , 130 µg·L -1 correspondent to 2.0 µM), cadmium (Cd 2+ , 15 µg·L -1 correspondent to 0.134 µM) and Tattoo fungicide (commercial formulation containing 45% of propamocarb-HCl (propyl-3-(dimethylamino) propylcarbamate and 55% of mancozeb, or Zn, Mn ethylenebis(dithiocarbamate) [-SCSNH(CH 2 ) 2 NHCSSMn-]n(Zn)m(Mn)], 91 µg L −1 ). Applied metal and Tattoo concentrations were based on their ecologically relevant concentrations for fresh water (US EPA, 2007) and previous experience with the same exposures effect on bivalve mollusks [8,9,11]. Mollusks were exposed during 14 days in 80 L tanks with aerated, softened tap water. Water and studied substances were renewed every two days. Mollusks were fed on lettuce before water changes. Mortality of snails was not observed during the experiment. Sampled tissues were stored at -40 °C. Each procedure of tissue preparation was carried out at 4 °C. Protein concentration in the supernatant (soluble protein) was measured by the method of Lowry et al. (1951) [21], using bovine serum albumin as protein standard. The absorbance values were measured on UV/Vis spectrophotometer "Lomo-56" (Russian Federation).
MTs were obtained as the thermostable proteins by size-exclusion chromatography on Sephadex G-50 for determining of the metal (Zn, Cu, Cd) concentrations [31]. Tissue samples were pooled and each pool was created from equal mass of aliquoted tissue (total mass 350 mg), and then homogenized in ice-cold 10 mM Tris-HCl buffer, pH 8.0, containing 10 mM 2-mercaptoethanol to prevent the MT from oxidation and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) to prevent protease activity. Despite the protease inhibitors do not appear to be required to recover metallothionein [19], the utilizing of PMSF is a common condition of the homogenate preparation and provides the avoiding of the low molecular weight products of the proteolysis in the thermostable supernatant.
This supernatant was incubated under the 85 °C for 5 min and subsequently centrifuged at 16,000 × g for 45 min at 4 °C. The obtained thermostable supernatant material was applied to a size-exclusion chromatography on Sephadex G-50 superfine column (1.5 × 50 cm) in the same buffer at a flow rate 0.33 ml·min -1 . Fractions (5 ml) were collected and analyzed for absorbance at 280 and 254 nm. Column calibration was achieved by applying a mixture of the appropriate protein standards: chymotrypsinogen (25.8 kDa), myoglobin (17.0 kDa), cytohrome c (12.3 kDa), ubiquitin (8.4 kDa), insulin chain B oxidized (3.5 kDa). MT-containing fractions were identified based on unique spectral features (comparative high density ratio D 254 /D 280 ), thermostability, and low molecular mass. The correspondent fractions were pooled (total 10 mL) for metal determination or anion-exchange chromatography.
The anion-exchange chromatography of the thermostable solution was carried out on a column (1.5 × 50 cm), packed with DEAE-cellulose. After removing of non-bound proteins by the column rinsing with 70 ml of buffer, elution was carried out in gradient mode with 0-1.0 M NaCl in a 10 mM Tris-HCl (pH 8.0) buffer containing 10 mM 2-mercaptoethanol and 1 mM 2-isopropanol at a flow rate of 0.5 ml·min -1 . Column calibration was achieved by applying standard metallothionein from rabbit liver. The fractions of each peak with high absorbance at 254 nm were pooled for the ultraviolet (UV) absorption spectra and the metals determination.
Metallothioneins' total protein concentration (MT-SH) was determined from thiols measure with DTNB after the ethanol/chloroform extraction from tissue [36]. The level of MT-SH was calculated assuming the relationship: 1 mol MT = 20 mol GSH and expressed as mg of MT per g of fresh tissue (FW).
The concentration of Cu, Zn and Cd was measured in samples of the weighed tissue (250 mg from each specimen). The samples were dried for 24 h at 105 °C, and then digested with 5 ml HNO 3 for 3 h at 105 °C under pressure and using acid-cleaned Teflon bomb. The content of Cu and Zn was evaluated by the atomic absorption spectrophotometry against certified standards on the spectrometer C-115, ("Lomo", Russian Federation), and Cd was analyzed by the graphite furnace atomic absorption spectrometer S-600 ("Selmi", Ukraine). Detection limits for metals were 0.1 µg·g -1 FW. The reliability of the metals measurements was assessed by analyzing ERM-CE 278 certified refe rence material; metal recovery was between 90 and 110%. Quality control was assessed by the method of Standard Addition [2]. Metal concentration in tissue and MT was expressed as µg·g -1 fresh weight (FW). MT metal-binding characteristics (MT-Me) was calculated from the concentrations of metals in the MT considering that one molecule of MT binds 7 Zn 2+ or Cd 2+ ions or 12 Cu + ions [25].
Thermostable proteins chemical analysis was carried out in triplicate on the joint sample from five individuals, and all other measurements were carried out in 8 specimens. A reliability of deviation of two series was calculated using the Student's t-test. Difference between group was considered reliable at P<0.05. The results were expressed as means ± standard deviation (S.D.). All statistical calculations were performed with Statistica v 12.0 and Excel for Windows-2000.

RESULTS AND DISCUSSION
From our previous experience, it is known that low weight cellular MT thiols belong to most sensitive biochemical indicators in L. stagnalis demonstrating a multiple variability of the metal composition and chromatographic forms under the environmental detrimental effects, and also, under the such nonspecific for this protein exposure as the effect of ionizing radiation [13,14]. In the current study, to attest their participation in metal detoxification and stress response, we evaluated total MT protein concentration (MT-SH), the level and composition of their metal-contained form (MT-Me) and their chromatographic characteristics.
Under size-exchange chromatography MT-containing peak was identified from low molecular mass (about 10 kDa), UV-spectrum with the typical for metal-thiolate clusters maximum at ~ 254 nm and absence of the peak at 280 nm, typical for the common proteins (Fig. 1A,C) [19,31]. The ion-exchange chromatography revealed two peaks corresponding to a distribution of the rabbit MT -MT-1 and MT-2 in the control group of snails. This behavior of the protein confirmed the purity and typical features of the MT [19]. The profile of MT under the ion-exchange chromatography was distorted at the exposures to metals and Tattoo, and the intermediate fraction, MT-2A, appeared (Fig. 1B). Typical UV-spectra of the metallothionein in the exposed snails were indicated according to the size-exclusion chromatography (Fig. 1C). Under the ion-exchange chromatography, the MT spectra were changed demonstrating a vulnerability of separate forms after a second step of purification (Fig. 1D).
The most expected response of mollusks at exposures to metals Cd, Zn, Cu is accumulation of correspondent metal in tissue of the digestive gland and its metallothionein [1]. Indeed, an exposure to metals caused an elevation of metal concentrations in a digestive gland (Fig. 2А,B,C). At the same time, concentration of effective metal was elevated only for Zn and Cd, whereas an exposure to Cu did not cause its excessive accumulation. The level of metals in a digestive gland was correspondent to control value at the exposure to Tattoo, The elevated level of Zn (by ~2−3 times) in tissue was the most specific response at the exposures to metals in comparison with the effect of thiocarbamate. The Tattoo per se is a source of Zn due to its formula. Therefore, the   [23]. In the present study, the level of Zn in the exposure to Tattoo was by 40 times lesser than in the exposure to the ionic form of Zn. Additionally, the responses to ionic and organic forms of metal could be different, as it was reported at the exposures of bivalve mollusk to ionic cobalt and Co 2+ -containing nanoscale polymeric complex [10]. Interestingly, the bivalve mollusk Anodonta cygnea demonstrated opposite responses at the same exposures conditions avoiding accumulation of metals in tissues [8,9]. Metal composition of the MT in snails was affected substantially (Fig. 2D,E,F). The levels of Cu and Cd were increased in the metallothionein at the exposures to Cu, Cd, Tattoo; the level of Zn was increased by the exposures to Cd and Tattoo. However, despite known preference of metallothionein in the Cd binding, only 16% of Cd in the tissue of snail was bound with the metallothionein at the correspondent exposure. In the study of Leung et al. (2003) [20], low level of Cd (of 0.01 µg/L) also did not change metal accumulation in the MT of the aquatic snails. Only the exposure to high concentration 1,000 µg/L of Cd during 10 days increased the level of MT-like proteins and the concentrations of metals (Cd, and also Ni and Cr). At the same time, there was no difference in Cu, Zn and Pb concentrations between the exposed and control groups.
Nevertheless, the total level of metal accumulation in the metallothionein was the highest at the exposures to Cd (level of Cu and Zn, and also, but much less Cd) and Tattoo (Cu, Zn and Cd). The concentration of metal-contained MT (MT-Me) was increased at these exposures by 2.2−2.6 times in comparison to control value and it was not changed at the exposure to Zn and Cu (Fig. 3B). However, MT accumulated low amount of Cu (~ 1-10 %) and Zn (~ 3-12 %) containing in tissue, whereas their part in Cd accumulation reached 84 % (at the exposure to Tattoo). A proportion of metal concentrations in the composition of MT (Zn:Cu:Cd) did not change prominently, therefore in all studied groups Zn was the main metal in composition of MT.

MT-SH/MT-ME
In the studies of R. Dallinger and colleagues [5,26,27], the selected accumulation of Cu and Cd in different MT forms of the Roman snail Helix pomatia under the experimental exposures [5,26,27] were proved. Mixed Cd, Cu-MT isoform has been also detected in the terrestrial pulmonate mollusk but in low abundance [18]. However, it was found that the metal intake in L. stagnalis has special features in comparison to the terrestrial Gastropoda. Although this snail possesses MT [14], it also binds ions of metals by thiol-contained peptides phytochelatins, and their level increased at the exposure to Cd [16]. Moreover, in the study of Niederwanger et al. (2017), MT of freshwater snail Biomphalaria glabrata lacked a clear metal-binding preference for Cu, Zn and Cd, that is clearly unprecedented in the Gastropoda MT. Absence of changes in MT gene transcription of B. glabrata after 21 days duration of Cd exposure (75 µg/L) confirmed a particular metal intake in aquatic snails [24]. Our results confirm low rate of the metallothionein involving in the metals binding in the aquatic snail.
Importantly, at the case of the exposure to Tattoo elevated metalation of metallothionein was not accompanied by an increased total metal concentration in tissue. Consequently, the metal-chelating thiocarbamate compound [7,30] caused a redistribution of metals within the tissue among different molecular targets. Similar increased participation of MT in metal binding at the exposure to Tattoo was observed in the bivalve mollusk [12].
Total concentration of metallothioneins (MT-SH) was increased by 3.6 times in all cases of metal exposures and much less (by 1.4 times) at the exposure to Tattoo comparative to control value (Fig. 3A). The exposure of L. stagnalis to extremely high concentration of Cd (50 mg L −1 ) during 20 days also caused an increase in total metallothionein concentration [6].
The calculated MT-SH/MT-Me concentration ratio (Fig. 3C) was increased substantially at exposures to metals: in Cu-and Zn-exposed groups by 3.5 times and in the Cd-exposed group -by 1.3 times than in control group. In a group exposed to Tattoo, it was lower than in the control. Indeed, at the exposures to metals, an increased level of the metallothionein was associated with the apoform of this protein, the same as in the bivalve mollusks [8], whereas, at the exposure to thiocarbamate the metal-binding function of metallothioneins was more evident. At increased MT concentration (7.6-fold) under the thiocarbamate action was reported in rat liver at 17 h after a single injection [34]. This effect on the MT was explained later as the dithiocarbamates inactivated reactive oxygen intermediates, chelate Zn(II) and synergistically augmented the induction of MT-1 mRNA levels via the antioxidant response element [29]. In current study, at low thiocarbamate concentration, the metallothionein synthesis was likely not targeted. However, a particular impact on the metal chelating was evident.
To summarize, common features of metallothionein responses in the aquatic mollusks can be noted as a partial saturation by metal exposures. Low level and vulnerability of the metallothioneins in snails confirms a finding of the R. Dallin ger and colleagues [5,18,24,26,27] concerning a particular metal sequestration in the aquatic snails.

CONCLUSION
In present study, all exposures to metals caused an elevation of concentration of metals (Zn, Cu or Cd) in tissues, whereas thiocarbamate fungicide did not affect it. The effect of metals and Tattoo on the levels of metallothionein metalation was opposite.
A distorted distribution of metals in tissue under the exposure to Tattoo needs more precise study of the initial targets of its effects.