Physiological and biochemical responses to caffeine and microplastics in Mytilus galloprovincialis.

Caffeine (Caff) is one of the most widely used substances in the human diet and a well-recognized drug. Its input into surface waters is remarkable, but biological effects on aquatic organisms are unclear, particularly in combination with pollutants of suspected modulatory activity, like microplastics (MP). The aim of this study was to reveal the impact on the marine mussel Mytilus galloprovincialis (Lamark, 1819) of Caff (20.0 µg L-1) in the environmentally relevant combination (Mix) with MP 1 mg L-1 (size 35-50 µm) after the exposure for 14 days. Untreated and exposed to Caff and MP separately groups were also examined. Cell viability and cell volume regulation in hemocytes and digestive cells, as well as the indexes of oxidative stress, glutathione (GSH/GSSG) and metallothioneins levels, and caspase-3 activity in digestive gland were assessed. MP and Mix reduced Mn-superoxide dismutase, catalase, and glutathione S-transferase activities and level of lipid peroxidation, but increased the digestive gland cell viability, GSH/GSSG ratio (by 1.4-1.5-fold), metallothioneins level and their Zn content, while Caff did not affect oxidative stress indexes and metallothionein-related Zn chelation. Protein carbonyls were not targeted in all exposures. The distinguishing feature of the Caff group was the decline (2-fold) in caspase-3 activity and low cell viability. The multi-stress effect of Mix was shown by the worsening of the volume regulation of digestive cells and confirmed by discriminant analysis of biochemical indexes. The special capabilities of M. galloprovincialis as a sentinel organism make it an excellent bio-indicator reflecting the multi-stress effects in sub-chronic exposures to potentially harmful substances. The identification of the modulation of individual effects in combined exposure increases the need to base monitoring programs on studies of multi-stress effects in sub-chronic exposures.

Interspecies comparison of selected pollution biomarkers in dreissenid spp. inhabiting pristine and moderately polluted sites.

Stress biomarkers, which can outline impacts of contaminants in aquatic biota at the biochemical level, are increasingly used as early warning tools in environmental monitoring. Reliable biomarker based assessment schemes, however, request appropriate knowledge of baseline levels of selected endpoints, and the potential influence of a range of natural influencing factors (both abiotic and biotic) as well. In this study, we examined the interspecies variability of various biomarkers (metallothioneins (MT), ethoxyresorufin-O-deethylase activity (EROD), lipid peroxidation (LPO), DNA strand breaks (DNA_sb), vitellogenin-like proteins (Vtg)) in Dreissena polymorpha and Dreissena bugensis inhabiting either pristine- or moderately impacted sites of Lake Balaton (Hungary). Levels of all biomarkers considered revealed low interspecies variability in the two dreissenid species at all sampling sites, with consistently higher (but statistically insignificant) values in Dreissena polymorpha. Levels of all biomarkers varied within the two investigated seasons, with significant influence of the reproduction cycle particularly on the levels of metallothioneins and vitellogenin-like proteins. Each biomarker considered was elevated by October, with significantly higher values in the mussels inhabiting harbours. Insignificant spatial and temporal variability in the general health indicators (condition index, total protein content) of dreissenids was observed, which, in parallel with evident rise in biomarker levels, apparently suggest that the anthropogenic impacts in harbours affect mussel fitness yet at sub organismal level. Our data might serve useful basis for future environmental monitoring surveys, especially in habitats where the progressive replacement of Dreissena polymorpha by Dreissena bugensis is taking place, as the interspecies variability in susceptibility to chemical stress of the two species is well comparable.

Interpopulational variability of molecular responses to ionizing radiation in freshwater bivalves Anodonta anatina (Unionidae).

Freshwater ecosystems are exposed to multiple anthropogenic stressors including chemical pollution and warming that can affect health of the resident organisms and their responses to novel challenges. We investigated the of in situ exposure history on molecular responses to a novel stressor, ionizing radiation, in unionid mollusks Anodonta anatina. Males from pristine (F-), agricultural (A-) sites and a cooling reservoir of a nuclear power plant (N-site) were exposed to acute low dose (2mGy) X-ray radiation followed by 14days of recovery (R-groups) or to control conditions (C-groups). Biomarkers of oxidative stress, geno-, cyto- and neurotoxicity were used to assess cellular injury and stress. Control group from the cooling reservoir (CN) had higher background levels of caspase-3 activity, metallothionein concentrations and nuclear lesions and lower levels of superoxide dismutase (SOD) and glutathione in the gills compared to other control groups (CF and CA). Irradiation induced cellular damage in mussels from all three sites including increased levels of nuclear lesions in hemocytes, depletion of caspase-3, suppression of superoxide dismutase and catalase activities, an increase of the lipid peroxidation and oxidized glutathione levels, as well as down-regulation of cholinesterase indicating neurotoxicity. The up-regulation of ethoxyresorufin-O-deethylase activity in the digestive gland and vitellogenin-like protein level in gonads were also found in radiation-exposed groups indicating feminization of males and disturbances of xenobiotic metabolism. The RA-group showed the greatest magnitude of radiation-induced stress responses compared to the other two groups. Overall, unionid mollusks, particularly those from a chronically polluted agricultural site, were highly sensitive to low-dose radiation (2mGy) indicating limitations of stress protection mechanisms to deal with multiple stressors.

Neonicotinoid insecticides inhibit cholinergic neurotransmission in a molluscan (Lymnaea stagnalis) nervous system.

Neonicotinoids are highly potent and selective systemic insecticides, but their widespread use also has a growing impact on non-target animals and contaminates the environment, including surface waters. We tested the neonicotinoid insecticides commercially available in Hungary (acetamiprid, Mospilan; imidacloprid, Kohinor; thiamethoxam, Actara; clothianidin, Apacs; thiacloprid, Calypso) on cholinergic synapses that exist between the VD4 and RPeD1 neurons in the central nervous system of the pond snail Lymnaea stagnalis. In the concentration range used (0.01-1 mg/ml), neither chemical acted as an acetylcholine (ACh) agonist; instead, both displayed antagonist activity, inhibiting the cholinergic excitatory components of the VD4-RPeD1 connection. Thiacloprid (0.01 mg/ml) blocked almost 90% of excitatory postsynaptic potentials (EPSPs), while the less effective thiamethoxam (0.1 mg/ml) reduced the synaptic responses by about 15%. The ACh-evoked membrane responses of the RPeD1 neuron were similarly inhibited by the neonicotinoids, confirming that the same ACh receptor (AChR) target was involved. We conclude that neonicotinoids act on nicotinergic acetylcholine receptors (nAChRs) in the snail CNS. This has been established previously in the insect CNS; however, our data indicate differences in the background mechanism or the nAChR binding site in the snail. Here, we provide the first results concerning neonicotinoid-related toxic effects on the neuronal connections in the molluscan nervous system. Aquatic animals, including molluscs, are at direct risk while facing contaminated surface waters, and snails may provide a suitable model for further studies of the behavioral/neuronal consequences of intoxication by neonicotinoids.

MANIFESTATIONS OF OXIDATIVE STRESS AND MOLECULAR DAMAGES IN OVARIAN CANCER TISSUE.

Indices of oxidative stress are recognized molecular markers and prognostic criteria for malignant transformation of tissue, but their value depends on the type of tumor and the stage of its development. The goal of this study was to clarify the relationship between the characteristics of the oxidative stress system including metal-associated ones and the cytotoxicity manifestations in neoplastically transformed human ovarian tissue. The highest level of Mn-superoxide dismutase activity (by 630%) and metallothionein protein (MT, 100%) has been estimated for the first time in malignant ovarian tissue compared to normal ovarian tissue. The researchers have also found a much higher level of oxy-radical formation (by 332%), a lower activity of catalase (by 49%) and a lower level of reduced glutathione (by 46%) and its redox index (0.84 versus 0.89 in the control) in tumor tissue. Under the relatively stable content of zinc, copper and cadmium in MTs, the content of zinc and especially copper in a form non-binding with MTs was significantly lower in the malignant tissue compared to normal one while the content of cadmium was higher. A discriminant analysis of all definable parameters revealed that the higher content of the products of oxidative destruction of proteins, lipids, fragmented DNA and the activity of cathepsin D, especially in its free form (by 235%), are the main characteristic signs of malignant ovarian tissue.

Habitat pollution and thermal regime modify molecular stress responses to elevated temperature in freshwater mussels (Anodonta anatina: Unionidae).

Elevated temperature and pollution are common stressors in freshwater ecosystems. We study cellular stress response to acute warming in Anodonta anatina (Unionidae) from sites with different thermal regimes and pollution levels: a pristine area and an agriculturally polluted site with normal temperature regimes (F and A, respectively) and a polluted site with elevated temperature (N) from the cooling pond of an electrical power plant. Animals were exposed to different temperatures for 14 days and stress response markers were measured in gills, digestive gland and hemocytes. Mussels from site N and A had elevated background levels of lactate dehydrogenase activity indicating higher reliance on anaerobic metabolism for ATP production and/or redox maintenance. Exposure to 25°C and 30°C induced oxidative stress (indicated by elevated levels of lipid peroxidation products) in digestive gland and gills of mussels from A and F sites, while in mussels from N sites elevated oxidative stress was only apparent at 30°C. Temperature-induced changes in levels of antioxidants (superoxide dismutase, metallothioneins and glutathione) were tissue- and population-specific. Acute warming led to destabilization of lysosomal membranes and increased frequencies of nuclear lesions in mussels from F and A sites but not in their counterparts from N site. Elevated temperature led to an increase in the frequency of micronuclei in hemocytes in mussels from F and A sites at 25°C and 30°C and in mussels from N site at 30°C. The mussels from N site also demonstrated better survival at elevated temperature (30°C) than their counterparts from the F and A sites. Taken together, these data indicate that long-term acclimation and/or adaptation of A. anatina to elevated temperatures result in increased thermotolerance and alleviate stress response to moderate temperature rise. In contrast, extreme warming (30°C) is harmful to mussels from all populations indicating limit to this induced thermotolerance.

[Trace elements storage peculiarities and metallothionein content in human thyroid gland under iodine deficiency euthyroid nodular goiter].

Accumulation of iodine and copper in the node, paranodular and contralateral (not affected tissue by node) tissues of thyroid gland in relation to the level of metal-binding proteins, potential antioxidants and oxidative changes in tissue was investigated. To assess the severity of the pathological process the molecular markers of cytotoxicity were used. The reduction of total iodine (by 19.5%), increase of inorganic iodine fraction (by 82.4%) and total copper content (twice) in paranodular and nodular tissues compared with contrlateral part have been established. Excess of copper in goitrous-changes tissue was partially accumulated in the metallothioneins. The level of metal-binding form of metallothioneins and reserve of free thiols of these proteins was higher two-three times and lower content of reduced glutathione in node-affected tissue compared to the contralateral part. Signs of cytotoxicity among them: higher cathepsine D free activity (up to 84.6% and 134.4% in paranodular tissue and node respectively) and higher level of DNA strand breaks in the node (up to 22.6%) were observed. In paranodular tissue the range of indices variability compared with parenchyma of contralateral part is shorter than in the node. Thus, under low level of iodine organification and high copper level in goitrous-modified tissue of thyroid gland metallothionein may provide a partial compensatory effect on prooxidative processes.

[Functions of metallothioneins and a system of antioxidant defense under the effect of Co- and Zn-containing nanocomposites on crucian carp (Carassius auratus gibelio)].

The effect of metal-nanocomposites (Me-NC) of cobalt and zinc (Co- and Zn-NC, correspondingly) synthecized on the basis of vinylpyrrolidone (PS) on the metal-accumulative proteins with antioxidant potential metallothioneins (MT) in crucian carp (Carassius auratus gibelio) was studied. Fish was subjected to the effect of Co-NC, Zn-NC, Co2+, Zn2+ or polymer carrier (PC) in the concentrations correspondent to 50 microg x Co/l or 100 microg x Zn/l during 14 days. It was shown that the MTs response is highly specific for the nature of metal, both in ion and Me-NC form: the effect of Co and Co-NC provoked the elevation of total MT concentration (MT-SH) and activation of antioxidant defence, whereas Zn and Zn-NC induced the decrease of the concentration of MT-SH and the inhibition of antioxidant defense. All the exposures provoked the decrease of the concentration of immunoreactive chelating MT form (MTi) and reduced glutathione, activation of anaerobiosys and Mn-superoxide dismutase, and also decrease of the concentration of proteins and lipids oxidative injury products. It was accompanied by the increase of the content of erythrocytes with nuclear abnormalities but did not cause the decrease of choline esterase activity. According to the rate of MT-SH and MTi concentrations, antioxidant potential of MTs is determined by its apoform. Our data indicate that partial biodegradation of Me-NC occurs in the organism of crucian carp.

[Population-related peculiarities of molecular stress-responsive systems of bivalve mollusk under the effect of tetrazine pesticide].

Tetrazine pesticides are widely used for the treatment of crops in most EU countries and USA. However, data about the effect of environmentally realistic concentrations of biocides on the molecular stress response system in non-target organisms are absent. The aim of our study was the comparison of adaptive capability of bivalve mollusk Anodonta cygnea from two populations under the effects of commercial pesticide Apollo in terms of biochemical parameters of the digestive gland. The differences between parameters of oxidative stress and glutathione transferase activity in specimens of control groups from clean (I group) and polluted (B group) areas have been shown. Under the effect of Apollo, the level of protein carbonyls and microsomal oxidation processes increased, and the level of metallothioneins and oxyradical formation decreased in the specimens from both populations. However, the treatment provoked the activation of antioxidant processes in the I group and their inhibition in B group. Potentially the injury of cellular thiols, glutathione and metallothioneins, seems to be key point of tetrazine pesticides toxicity.

[Evaluation of biological effects of cobalt-nanocomposites with the use of biochemical markers of bivalve mollusk Anodonta cygnea].

Intensive implementation of nanomaterials requires development of novel methods for evaluation of their potential ecotoxicity. The aim of our study was to identify specific characteristics of the effect of cobalt-nanocomposite (Co-NC) on the molecular stress-responsive system in the digestive gland of bivalve mollusk Anodonta cygnea. Nanocomposite was synthesized by mixing alcohol solution of copolymer N-vinylpirrolidone, 5-(tret-butylperoxy)-5-methyl-1-hexene-3-yne and dimethylaminoethylmetacrylate and cobalt (II) chloride. After 14 days of the mollusk exposure in the presence of Co-NC, CoCl, or corresponding polymer substance it was shown that the Co-NC, in contrast to other agents, does not cause an oxidative stress due to the superoxide dismutase activity, metallotioneins (MTs) level, glutathione redox index and oxyradical production. Multivariate analysis confirmed specific features of the Co-NC's effect related to an enhanced expression of MTs, while CoCl2 activated lactate dehydrogenate and oxyradical production, and polymer substance enhanced glutathione transferase activity.

[Metal-binding functions and antioxidant properties in human thyroid gland under iodine deficient nodular colloidal goiter].

Copper and zinc levels in the tissue of thyroid gland (TG) and their metal-binding proteins metallothioneins (MT) as well as state of the antioxidant system in persons that had no thyroid disease and patients with endemic iodine deficiency nodular colloidal goiter has been investigated. In the patients with thyroid disease, oxidative damage was indicated despite elevated levels of MT-SH and glutathione, and elevated copper and decreased zinc concentration in TG tissue. MTs partly bound the excess of copper but its concentration in the unbound to MT form was two-fold compared to the control value.

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.

[The effect of pesticide acetamiprid on biochemical markers in tissues of fresh water bivalve mussels Anodonta cygnea L. (Unionidae)].

The effects of ecological real concentration of pesticide "Mospilan" (active substance - acetamiprid) on the status of oxidative stress biomarkers, neurotoxicity and metallothioneins' (MTs) characteristics of freshwater bivalve mussels Anodonta cygnea were investigated. The prooxidative changes (decrease of superoxide dismutase activity, reduced glutathione content, increase of content of lipid and proteins peroxidation products) and decrease of the MTs content in gills and especially in digestive gland in the experimental group were reflected, whereas acetylcholinesterase activity was not changed. The effect of acetamiprid induces the appearance of additional chromatographic MTs form.

[Effect of zinc ions, manganese and lead ions on the thermostable proteins of carp liver]. / Vplyv ioniv tsynku, marhantsiu ta svyntsiu na termostabil'ni bilky pechinky koropa.

Effect of comparatively low concentration of the zinc, manganese, lead ions (0.1, 0.13 and 0.01 mg/l respectively) on the thermostable proteins (TP) of the carp liver after fish incubation for 14 days was studied. It is revealed that about 30 of zinc, 40% of manganese and 6% of lead were bound in the TP solution. The results of chromatography on Sephadex G-75 show, that the metallothionein-like proteins (MT, M-6 kDa) were the primary metal-binding proteins among the TP for zinc and manganese, and the heavy fraction of TP (M-60 kDa)--for lead. Under the action of each of the metallions its content in the TP is significantly increased. The distribution of metals among TP compounds is similar to that in the control. The metals action causes specific changes of the heavy fraction properties (UV spectrum, SH-groups content). The properties of MT do not change significantly under the control value.

[Regulatory mechanisms of oxygen transport function of hemoglobin in irradiated animals]. / Issledovanie mekhanizmov reguliatsii kislorodtransportnoi funktsii gemoglobina u obluchennykh zhivotnykh.

The oxygen affinity of hemoglobin of both common and 2,3-diphosphoglycerate concentration-purified erythrocyte hemolysates, hemoglobin stability, 2,3-diphosphoglycerate concentration in erythrocytes, morphological blood content were studied in rabbits irradiated with X-rays in a dose of 0.155 C/kg. An increase in the oxygen affinity of common hemolysate hemoglobin at a higher 2,3-diphosphoglycerate concentration on the 1st, 4th day after irradiation and a decrease in the oxygen affinity of both common and purified hemolysate hemoglobin on the 18th-30th day after irradiation were observed. A conclusion is made that disturbance of the hemoglobin function control by 2,3-diphosphoglycerate in irradiated animals is determined by alteration of protein physicochemical properties.

Secretory IgA against enterotoxins in breast-milk.

A pool of colostrum from Guatemalan mothers (Guatemalan colostrum)) obtained 2-4 days post partum inhibited the induced fluid accumulation in rabbit ileal loops when incubated with Vibrio cholerae or Escherichia coli enterotoxin. There was a linear relationship between the quantity of colostrum used and the protection achieved. Pools of Guatemalan breast-milk obtained 15-30 days post partum and North American breast-milk had the same effect when tested with E. coli and V. cholerae enterotoxins, respectively. The antitoxic activity of a given pool correlated with its IgA content but not with the concentration of IgG or IgM. Guatemalan colostrum globulins were precipitated by ammonium sulphate. The globulins were filtered through a 'Biogel A5' column and fractions obtained. When tested in rabbit ileal loops the antienterotoxin activity in these fractions closely paralleled their IgA but not their detectable IgG or IgM content. We hypothesise that IgA antibody to enterotoxin, present in breast-milk of normal mothers, is probably a manifestation of natural immunity. The passive transfer of these antibodies to the infant may explain why breast-milk prevents E. coli diarrhoea in the neonate.