Effects of hypoxia and reoxygenation on intermediary metabolite homeostasis of marine bivalves Mytilus edulis and Crassostrea gigas.

Marine benthic invertebrates are frequently exposed to fluctuating oxygen levels resulting in hypoxia-reoxygenation (H/R) stress in the intertidal, estuarine and shallow coastal habitats. H/R stress can strongly affect the organisms' physiological performance due to the negative shifts in bioenergetics and redox balance. H/R stress commonly leads to the depletion of energy substrates and accumulation of anaerobic end products, but the effects of H/R stress on the homeostasis of the intermediate nitrogenous compounds are not well understood. We studied the effects of the short-term and long-term hypoxia (1 and 6 days, respectively) and subsequent reoxygenation on the metabolite profiles of free amino acids (FAAs), as well as the intermediates of the urea cycle and purine metabolism in two species of hypoxia-tolerant intertidal bivalves, the blue mussels Mytilus edulis and the Pacific oysters Crassostrea gigas. Accumulation of succinate was assessed to determine the role of anaerobiosis in the metabolic responses to H/R stress. Our study showed that the more hypoxia-tolerant of the two studied species (C. gigas) had lower rate of succinate accumulation during hypoxia (indicating stronger metabolic rate suppression) and was better able to maintain the homeostasis of nitrogenous intermediates during H/R stress compared with the less hypoxia-tolerant M. edulis. Furthermore, analysis of the metabolite profiles indicate that the oysters maintain high levels of cytoprotective compounds (such as taurine and GABA), accumulate lower levels of potential prooxidants (such as succinate and hypoxanthine) and experience less damage to oxidation-prone thiol-containing amino acids such as cysteine, homocysteine and methionine during hypoxia and reoxygenation compared with the blue mussels. This study indicates a potentially important role of intermediate metabolite homeostasis in the tolerance to prolonged hypoxia and H/R stress in marine organisms and opens avenue for further testing of this hypothesis in a broader comparative framework.

The effects of ZnO nanostructures of different morphology on bioenergetics and stress response biomarkers of the blue mussels Mytilus edulis.

Biofouling causes massive economical losses in the maritime sector creating an urgent need for effective and ecologically non-harmful antifouling materials. Zinc oxide (ZnO) nanorod coatings show promise as an antifouling material; however, the toxicity of ZnO nanorods to marine organisms is not known. We compared the toxicity of suspended ZnO nanorods (NR) with that of ZnO nanoparticles (NP) and ionic Zn2+ in a marine bivalve Mytilus edulis exposed for two weeks to 10 or 100 µg Zn L-1 of ZnO NPs, NRs or Zn2+, or to immobilized NRs. The multi-biomarker assessment included bioenergetics markers (tissue energy reserves, activity of mitochondrial electron transport system and autophagic enzymes), expression of apoptotic and inflammatory genes, and general stress biomarkers (oxidative lesions, lysosomal membrane stability and metallothionein expression). Exposure to ZnO NPs, NRs and Zn2+ caused accumulation of oxidative lesions in proteins and lipids, stimulated autophagy, and led to lysosomal membrane destabilization indicating toxicity. However, these responses were not specific for the form of Zn (NPs, NR or Zn2+) and showed no monotonous increase with increasing Zn concentrations in the experimental exposures. No major disturbance of the energy status was found in the mussels exposed to ZnO NPs, NRs, or Zn2+. Exposure to ZnO NPs and NRs led to a strong induction of apoptosis- and inflammation-related genes, which was not seen in Zn2+ exposures. Based on the integrated biomarker response, the overall toxicity as well as the pro-apoptotic and pro-inflammatory action was stronger in ZnO NPs compared with the NRs. Given the stability of ZnO NR coatings and the relatively low toxicity of suspended ZnO NR, ZnO NR coating might be considered a promising low-toxicity material for antifouling paints.

Bioenergetic responses of freshwater mussels Unio tumidus to the combined effects of nano-ZnO and temperature regime.

Bivalves from the cooling reservoirs of electrical power plants (PP) are exposed to the chronic heating and chemical pollution making them a suitable model to study the combined effects of these stressors. We investigated the effect of in situ exposures to chemical and thermal pollution in the PP cooling ponds on the metabolic responses of unionid bivalves (Unio tumidus) to a novel widespread pollutant, ZnO nanoparticles (nZnO). Male U. tumidus from the reservoirs of Dobrotvir and Burshtyn PPs (DPP and BPP) were maintained in clean water at 18 °C, or exposed for 14 days to one of the following conditions: nZnO (3.1 µM) or Zn2+ (3.1 µM, a positive control for Zn impacts) at 18 °C, elevated temperature (T, 25 °C), or nZnO at 25 °C (nZnO + T). Baseline levels of glycogen, lipids and ATP were similar in the two studied populations, whereas the levels of proteins, lactate/pyruvate ratio (L/P) and extralysosomal cathepsin D level were higher in the tissues of BPP mussels. The levels of glycogen and glucose declined in most experimental exposures indicating elevated energy demand except for a slight increase in the digestive gland of warming-exposed BPP mussels and in the gills of the nZnO + T-exposed DPP-mussels. Experimental exposures stimulated cathepsin D activity likely reflecting onset of autophagic processes to compensate for stress-induced energy demand. No depletion of ATP in Zn-containing exposures was observed indicating that the cellular metabolic adjustments were sufficient for such compensation. Unexpectedly, experimental warming mitigated most metabolic responses to nZnO in co-exposures. Our data thus indicate that metabolic effects of nZnO strongly depend on the environmental context of the mussels (such as temperature and acclimation history) which must be taken into account for the molecular and cellular biomarker-based assessment of the nanoparticle effects in the field.

Detoxification and cellular stress responses of unionid mussels Unio tumidus from two cooling ponds to combined nano-ZnO and temperature stress.

Bivalve mollusks from the cooling reservoirs of fuel power plants (PP) are acclimated to the chronic heating and chemical pollution. We investigated stress responses of the mussels from these ponds to determine their tolerance to novel environmental pollutant, zinc oxide nanoparticles (nZnO). Male Unio tumidus from the reservoirs of Dobrotvir and Burschtyn PPs (DPP and BPP), Ukraine were exposed for 14 days to nZnO (3.1 µM), Zn2+ (3.1 µM) at 18 °C, elevated temperature (T, 25 °C), or nZnO at 25 °C (nZnO + T). Control groups were held at 18 °C. Zn-containing exposures resulted in the elevated concentrations of total and Zn-bound metallothionein (MT and Zn-MT) in the digestive gland, an increase in the levels of non-metalated MT (up to 5 times) and alkali-labile phosphates and lysosomal membrane destabilization in hemocytes. A common signature of nZnO exposures was modulation of the multixenobiotic-resistance protein activity (a decrease in the digestive gland and increase in the gills). The origin of population strongly affected the cellular stress responses of mussels. DPP-mussels showed depletion of caspase-3 in the digestive gland and up-regulation of HSP70, HSP72 and HSP60 levels in the gill during most exposures, whereas in the BPP-mussels caspase-3 was up-regulated and HSPs either downregulated or maintained stable. BPP-mussels were less adapted to heating shown by a glutathione depletion at elevated temperature (25 °C). Comparison with the earlier studies on mussels from pristine habitats show that an integrative 'eco-exposome'-based approach is useful for the forecast of the biological responses to novel adverse effects on aquatic organisms.

Vulnerability of marsh frog Pelophylax ridibundus to the typical wastewater effluents ibuprofen, triclosan and estrone, detected by multi-biomarker approach.

Pharmaceutical and personal care products (PPCPs) are the environmental pollutants of growing concern. The aim of this study was to indicate the effects of typical PPCPs on the marsh frog Pelophylax ridibundus. We treated male frogs with waterborne ibuprofen (IBU, 250ng·L-1), triclosan (TCS, 500ng·L-1), or estrone (E1, 100ng·L-1) for 14days. Common vulnerability of the frogs was detected from dramatic decrease of Zn, total and metalated metallothionein (MT) concentrations, Zn/Cu ratio, the elevation of activity of glutathione-S-transferase, cathepsin D and DNA instability in the liver, the depletion of cholinesterase in the brain and cortisol in the blood plasma in all exposures. Nevertheless, lipofuscin concentration in the liver was always decreased. The groups were best distinguished by cytochrome P450 (CYP450) activity determined by ELISA. The exposure to IBU caused lesser damage, but elevated the levels of oxyradicals and glutathione (GSH and GSSG) and lysosomal membrane instability. Exposures to TCS and E1 provoked the endocrine disturbance (increased levels of vitellogenin and thyrotropin in blood plasma), decreased lactate dehydrogenase activity and increased level of pyruvate in the liver. TCS caused the increase of GSSG by 7.3 times and lactate levels. Only E1 lead to decrease of deiodinase activity in the liver, activation of CYP450 and caspase-3 and efflux of cathepsin D from lysosomes. Spectrophotometric and ELISA assays of MTs and CYP450 gave distinct results in E1-group. Broad disruption of the hormonal pathways caused by E1 could be of concern for the health status of frogs in their habitats.

Biomineralization-related specialization of hemocytes and mantle tissues of the Pacific oyster Crassostrea gigas.

The molluscan exoskeleton (shell) plays multiple important roles including structural support, protection from predators and stressors, and physiological homeostasis. Shell formation is a tightly regulated biological process that allows molluscs to build their shells even in environments unfavorable for mineral precipitation. Outer mantle edge epithelial cells (OME) and hemocytes were implicated in this process; however, the exact functions of these cell types in biomineralization are not clear. Pacific oysters (Crassostrea gigas) were used to study differences in the expression profiles of selected biomineralization-related genes in hemocytes and mantle cells, and the functional characteristics of hemocytes such as adhesion, motility and phagocytosis. The specialized role of OME in shell formation was supported by high expression levels of the extracellular matrix (ECM) related and cell-cell interaction genes. Density gradient separation of hemocytes revealed distinct phenotypes based on the cell morphology, gene expression patterns, motility and adhesion characteristics. These hemocyte fractions can be categorized into two functional groups, i.e. biomineralization and immune response cells. Gene expression profiles of the putative biomineralizing hemocytes indicate that in addition to their proposed role in mineral transport, hemocytes also contribute to the formation of the ECM, thus challenging the current paradigm of the mantle as the sole source of the ECM for shell formation. Our findings corroborate the specialized roles of hemocytes and the OME in biomineralization and emphasize complexity of the biological controls over shell formation in bivalves.

Long-Term Acclimation to Different Thermal Regimes Affects Molecular Responses to Heat Stress in a Freshwater Clam Corbicula Fluminea.

Global climate change (GCC) can negatively affect freshwater ecosystems. However, the degree to which freshwater populations can acclimate to long-term warming and the underlying molecular mechanisms are not yet fully understood. We used the cooling water discharge (CWD) area of a power plant as a model for long-term warming. Survival and molecular stress responses (expression of molecular chaperones, antioxidants, bioenergetic and protein synthesis biomarkers) to experimental warming (20-41 °C, +1.5 °C per day) were assessed in invasive clams Corbicula fluminea from two pristine populations and a CWD population. CWD clams had considerably higher (by ~8-12 °C) lethal temperature thresholds than clams from the pristine areas. High thermal tolerance of CWD clams was associated with overexpression of heat shock proteins HSP70, HSP90 and HSP60 and activation of protein synthesis at 38 °C. Heat shock response was prioritized over the oxidative stress response resulting in accumulation of oxidative lesions and ubiquitinated proteins during heat stress in CWD clams. Future studies should determine whether the increase in thermal tolerance in CWD clams are due to genetic adaptation and/or phenotypic plasticity. Overall, our findings indicate that C. fluminea has potential to survive and increase its invasive range during warming such as expected during GCC.

Diversity of the molecular responses to separate wastewater effluents in freshwater mussels.

The environmental safety of pharmaceutical and personal care products (PPCPs) requires a crucial examination. The aim of this study was to evaluate the responses of biomarkers of stress and toxicity in freshwater mussels to the effect of commonly found PPCPs in wastewater. We treated male mussels Unio tumidus, from an undisturbed site with ibuprofen (IBU, 250 ng L(-1)), triclosan (TCS, 500 ng L(-1)), or estrone (E1, 100 ng L(-1)) for 14 days. Untreated mussels from this site (C) and mussels inhabiting a polluted area (P) were also examined after a similar time of being kept in the laboratory. The consequences of chronic exposure of the mussels in the P-group were reflected in elevated concentrations of oxyradicals (1.4 times), oxidized glutathione (4.3 times), lipofuscin (2.2 times), and DNA-strand breaks in the digestive gland (DG) in comparison to the C-group, higher levels of caspase-3 activity in the DG, and vitellogenin-like proteins in gonads among all studied groups. Exposed mussels demonstrated some common responses with mussels in the P-group: elevated levels of lactate/pyruvate ratio, lipofuscin (IBU and E1), DNA fragmentation (TCS and E1), and caspase-3 activity (TCS and E1). Exposed to PPCPs mussels also showed elevation of ethoxyresorufin-O-deethylase and/or glutathione-S-transferase activity in the DG and a decrease in lysosomal stability in hemocytes (TCS and E1). The TCS group was distinguished by having the highest level of DNA-fragmentation and the lowest concentrations of total glutathione, oxyradicals, lipofuscin, pyruvate, and lactate, reflecting total metabolic depression. These results show that selected PPCPs at low concentrations alter a variety of physiological processes in this animal model system.

In situ exposure history modulates the molecular responses to carbamate fungicide Tattoo in bivalve mollusk.

The aim of the present study was the investigation of the effect of in situ exposure history on the responses of freshwater mussels to thiocarbamate fungicide. Male bivalve mollusks Anodonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to fungicide Tattoo (mixture of propamocarb and mancozeb, 91 µg L(-1)). When unexposed mussels were compared, chronic effect of toxic environment in site A was confirmed by oxidative stress indices (high levels of superoxide dismutase and catalase activities, lipid peroxidation, protein carbonyls and oxyradical production, low level of total glutathione (GSH)), genotoxicity (high levels of DNA-strand breaks and caspase-3 activity in digestive gland), and cytotoxicity (low lysosomal membrane stability in hemocytes), elevated vitellogenin-like proteins (Vtg-LP) concentration in gonads, high levels of Cu, Zn, Cd, metallothionein (MT)-bound metals (MT-Me) and MT-related thiol (MT-SH), and low ethoxyresorufin-O-deethylase (EROD) activity in digestive gland. The major differences in the responses of the two exposed groups were related to antioxidant defense and MT: in the group A, prominent oxidative stress response with the participation of MT-SH and GSH in the gills, EROD activation, but decrease of MT-Me level was shown, whereas in group F exposure provoked the elevation of MT-Me, caspase-3 and Vtg-LP values. Carbamate did not cause cholinesterase depletion and cytotoxicity. However, genotoxic and pro-oxidant effects (increased levels of hemocytes with micronuclei and nuclear abnormalities, DNA-strand breaks and oxyradical in digestive gland), were common responses for both the exposed groups.

Effect of in situ exposure history on the molecular responses of freshwater bivalve Anodonta anatina (Unionidae) to trace metals.

The goal of the study was to assess the adequacy of molecular responses in mollusks in relation to their in situ exposure history. Freshwater male bivalve mollusks Anadonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to copper (Cu(2+), 10 µg L(-1)), zinc (Zn(2+), 130 µg L(-1)) or cadmium (Cd(2+), 15 µg L(-1)). The comparison of two control groups showed that the specimens from site A had higher levels of Cu, Zn and Cd and metallothionein (measured both through metal (MT-Me), and protein (MT-SH) levels) in the tissues. Cytotoxicity (low lysosomal membrane stability), low glutathione level, high antioxidant and apoptotic enzymes activities, lipid and protein oxidative injury, depletion of ethoxyresorufin-O-deethylase (EROD) in digestive gland, high vitellogenin-like protein (Vtg-LP) concentration in gonads confirmed the effect of toxic environment on this group. Exposures provoked increased number of hemocytes with micronuclei (by 100-500%) and nuclear abnormalities (by 50-400%) (genotoxicity), elevation of caspase-3 (in 1.5-10 times) and/or Vtg-LP (by 70-310%) levels in all groups. However, the responses were strongly dependent on the origin of mussels. Exposed mussels from site F demonstrated typical for the effect of toxic metals elevation of MT-SH (by 100-380%) and MT-Me (up to seven times) levels and accumulation of metals (with a few exceptions) in the tissues. Conversely, in the mussels inhabiting site A, exposures caused the decrease of metal (by 37% for Cu, by 62% for Zn, by 50% for Cd), MT-SH (by 68% in ZnA group) and MT-Me (by 50-68%) levels. That was accompanied with increase of cytotoxicity and EROD activity (by 144-240%). High level of protein carbonyls was the distinguished feature of all groups from site A. Hence, despite high efficiency of metal detoxification and oxidative stress responses in the mussels, in the specimens from spontaneously polluted site they were impaired.

Main partitioning criteria for the characterization of the health status in the freshwater mussel Anodonta cygnea from spontaneously polluted area in Western Ukraine.

The aim of this study was to appreciate the consequences of spontaneous human activity for freshwater mollusks in the generally ecologically sustainable area in Western Ukraine. For this, bivalve mollusk, Anodonta cygnea, at three sites, with mixed agricultural and municipal activities (A), close to a municipal water inlet (F) and the cooling pond of a nuclear power plant (N), were studied in spring, summer, and autumn. The set of parameters included the characteristics of oxidative stress (activity of catalase (CAT), levels of protein carbonyls (PC)), levels of reduced and oxidized glutathione (GSH, GSSG, respectively), activities of lactate dehydrogenase (LD), cholinesterase (ChE), ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST) in the digestive gland, and concentrations of vitellogenin-like proteins (Vtg-LP) in gonads and also morphological indices. Although the discriminant functional analysis confirmed the general seasonal regularities for studied groups, it allowed to discriminate between sites (P < 0.05). At site A, oxidative stress; high levels of LD, EROD, and GST; and low levels of ChE and condition factor were reflected. This demonstrated the sensitivity of mussels to constant effect of mixed pollution. At site N, oxidative injury was shown that might be explained by the constantly high temperature. At site F, abrupt elevations of Vtg-LP and EROD levels in autumn were probably related to an emergency situation on the nearby dump. So, both chronic and temporal environmental effects were reflected by a set of markers in mollusk. The classification and regression tree (CART) algorithm selected GSH and PC in the digestive gland and Vtg-LP as partitioning criteria for the characterization of mussel health status. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Population-related molecular responses on the effect of pesticides in Carassius auratus gibelio.

The aim of this study was to evaluate population-related peculiarities of the adaptive responses of Carassius auratus gibelio. In order to do this, male specimens from polluted (B) and clean (Z) sites were exposed to commercial pesticides thiocarbamate Tatoo (9.1 µg·L(-1)and 91 µg·L(-1)) or tetrazine Apollo (2 µg·L(-1) and 10 µg·L(-1)) during fourteen days. The control fish from site B was distinguished by weakness of antioxidant defence (measured from superoxide dismutase and catalase activities, redox index of glutathione (GSH), superoxide anion (O(2)) and lipid peroxidation levels), imbalance of the concentrations of protein metallothionein (MT-SH) and MT-related metals (MT-Me) and neurotoxicity. Differences in glutathione-S-transferase activity in the liver and vitellogenin-like proteins in the serum were also showed between B and Z control groups. Common effects of pesticides were related to a decrease in GSH, an increase in O(2) production, ethoxyresorufin O-deethylase activity and hepatosomatic index. Apollo provoked particular elevation of MT-SH/MT-Me ratio. Population-related difference in the response was the activation of antioxidant defence in fish from site B and its inhibition in fish from site Z. The genotoxic effect of exposures was more expressed in fish from site B. Principal component analysis combine all exposed groups from site Z and control group from site B in one set, and separated each exposed group from site B. The main distinguishing index of each population selected by classification and regression tree analysis was MT-SH.

Various responses to copper and manganese exposure of Carassius auratus gibelio from two populations.

The aim of this study was to compare the effect of pro-oxidants copper (Cu(2+), 0.005 and 0.050mg L(-1)) or manganese (Mn(2+), 0.17 and 1.7mg L(-1)) on Carassius auratus gibelio from polluted (B) and unpolluted (Z) sites after exposure for fourteen days. Fish from site B showed high levels of lipid peroxidation (TBARS concentration), lower levels of metallothionein (MT)-related metal, total glutathione (GSH), its redox index, superoxide dismutase (Cu,Zn- and Mn-SOD), glutathione-S-transferase (GST) and cholinesterase (ChE) activities and also higher MT-related thiol concentration in the liver and gills. A common effect of exposure was related to genotoxicity, a decrease in GSH and an increase in microsomal ethoxyresorufin O-deethylase activity in the liver. However, the systems of oxidative stress and biotransformation were more efficient in fish from the polluted site, while the responsivity of MTs in this fish was impaired. Principle Component Analysis separated the subgroups from the unpolluted site and fish loaded by lesser concentrations of metals on the one side, and fish from the polluted site jointly with fish exposed to higher concentrations of metals on the other side. The main distinguishing indices of sites and exposures selected by classification and regression tree (CART) analysis were MT characteristics and genotoxicity.

Variability of responses in the crucian carp Carassius carassius from two Ukrainian ponds determined by multi-marker approach.

The aim of this study was to evaluate the health status of the comparatively tolerant fish Carassius carassius over three seasons in an area characterized by spontaneous human activities. Sites near the springs of a river (site Z) and downstream of a river (site B) in Western Ukraine were selected. According to the centroid grouping analysis, the biochemical and morphological indices allowed the fish to be distinguished according to season more than to site. The level of nuclear abnormalities was low in fish from both sites. However C. carassius inhabiting site B showed a lower metal-binding capacity of MTs in relation to fish from site Z. This was combined with high levels of MT protein (particularly in the liver), and reduced glutathione (GSH) and redox state of GSH (particularly in the gills), which might confer some advantages to fish inhabiting this site. The levels of ethoxyresorufin-O-deethylase, glutathione-S-transferase, cholinesterase and vitellogenin-like proteins indicated significant but intermittent inter-site differences. In summer, oxidative damage due to a high level of lipid peroxidation, and low superoxide dismutase and catalase activities was observed in fish from site B, and in autumn, it was observed in the gills of fish from site Z. The relationship between MT protein levels and antioxidant defense and the lack of a positive relationship between MT levels and their metal-binding capacity was confirmed by principal component analysis.

Vulnerability of biomarkers in the indigenous mollusk Anodonta cygnea to spontaneous pollution in a transition country.

The aim of this study was to estimate the sensitivity of biomarkers of stress and exposure in the bivalve mollusk Anodonta cygnea to spontaneous anthropogenic activities typical for the Western Ukraine. Three sites were examined during spring, summer and autumn: an agricultural site (A); the cooling pond of nuclear power plant (N) and a forestry close to the municipal water inlet (F). Common temporal changes of a battery of biochemical markers in the gills and hemolymph and morphological characteristics were shown by discriminant functional analysis. Classification trees built on the basis of the screened biomarkers demonstrated persistent peculiarities at each site: genotoxicity (nuclear abnormalities) at site A and endocrine disruption (high levels of vitellogenin-like proteins (Vtg-LP) in hemolymph) at site F. Interim local effects were best characterized by metallothionein (MT) concentrations, lipid peroxidation (LPO), activities of glutathione S-transferase and lactate dehydrogenase, and the conditional index of the gills. In autumn, the mollusks from the three sites revealed the highest differences in pollution status: the activation of antioxidant defense and cholinesterase were typical for site A, highest levels of MT related to high levels of Cu and Cd in the water at site B, and a steep increase in the level of Vtg-LP and the decrease of lysosomal membrane stability were recorded at the site selected as reference (F). The biomarker alterations recorded at site F were later related to an emergency event at the municipal dump located nearby. Thus, our case study demonstrated the reliability of using biomarkers of exposure to assess both long-term and accidental environmental pollution loads.

Function of metallothioneins in carp Cyprinus carpio from two field sites in Western Ukraine.

The aim of the present study was to elucidate the seasonal and spatial regularity of the properties of metallothioneins (MT) from the liver and gills of carp Cyprinus carpio L. in rural (R) and industrial (I) sites in Western Ukraine. The MT is represented by two chromatographic forms, the features of which exhibit seasonal rather than spatial dependence. The pronounced differences between the sites were due to the lower levels of Zn in the liver and the higher levels of Zn in MT of carp from site I, providing evidence of the higher overall anthropogenic impact here that leads to the distortion of this essential metal accumulation and to the activation of metal-binding function of MT. In spring, higher levels of Cu and Cd in MT and in the tissues were reflected at site R probably as the result of the permitting pollution here. The principal component analysis demonstrated the correlation of MT-bound metal levels to their levels in water and the absence of such relation for general tissue metal levels for Zn.

Multi-biomarkers approach in different organs of Anodonta cygnea from the Dnister Basin (Ukraine).

The aim of this study was to compare environmental quality in two sites in western Ukraine-rural (R) and urbanized (U)-with the usage of the resident bivalve mollusk Anodonta cygnea. The study was realized during three seasons. The metal uptake and a set of biochemical markers were determined. For each season, Cd and metallothioneins (MTs) contents in the digestive gland and gills of the mollusc were higher at the U site, reflecting its chronic pollution. The oxidative stress in the mollusk was observed at the U site during spring and at the R site during summer and autumn according to the differences in Mn-superoxide dismutase and catalase activities, O (2) (*-) production, lipid peroxidation, and glutathione levels. The elevated vitellogenin-like protein levels in the hemolymph and the ethoxyresorufin-O-deethylase activity in the digestive gland in summer-autumn suggested pollutions by organic substances at the R site. The acetylcholinesterase activity was similar in both groups. The centroid grouping analysis of biomarkers and morphological and water indexes demonstrated the clear differentiation of general response in each group in spring and, at the R site, in summer and autumn but its similarity at the U site in summer and autumn.

Responses of biochemical markers in carp Cyprinus carpio from two field sites in Western Ukraine.

The aim of this study was to compare environmental quality in two sites of the river in Western Ukraine, rural (R) and industrial (I) during three seasons via a set of biochemical markers in carp Cyprinus carpio L. Upon comparing the values of the I-site with those of the R-site, we found that Mn- and Cu, Zn-superoxide dismutase (SOD) activities decreased and O(.) production increased; metallothionein (MT) and glutathione levels increased in most cases, and acetylcholinesterase (AChE) activity decreased in summer. This confirms our hypothesis about continuous environmental press at the I-site. The higher activity of catalase and lipid peroxidation (TBARS), as well as the increased levels of vitellogenin-like proteins at the R-site, compare to the I-site in spring reflects the permitting effect of agricultural discharges. According to the results of PCA, the most sensitive biomarkers of pollution are MT, TBARS, and AChE in liver.

Seasonal and spatial comparison of metallothioneins in frog Rana ridibunda from feral populations.

The aim of this study was to elucidate the seasonal and spatial regularity of the properties of metallothioneins (MTs) from the liver and kidney of the frog Rana ridibunda in rural (R) and urban (U) sites in Western Ukraine. This allowed examination of their possibility use in biomonitoring of environmental quality. The positive correlation for Zn and negative correlation for Cu were reflected between their content in the liver and MTs. The content of MTs was higher in summer compared to other seasons and also at the U site compare to the R site. MTs had been comprised of two chromatographic forms (MT-1 and MT-2/MT-2a), with lesser and variable MT-2/2a in frogs from the U site, particularly in the kidney. MTs accumulated about 75% of Cd in the liver. In summary, the ability MTs to elevate content as a stress response, together with the sensitivity of MT-2, may be explored to understand the health status of the frog in each season, reflecting the higher overall anthropogenic impact at the U site.