Parameters of the antioxidant defence system and heavymetal concentrations in the visceral mass of selected snail and musselspecies from the Danube, Tisa and Velika Morava rivers

Slatkovodni mekušci su dobri model organizmi za proučavanje uticaja faktora sredine u biomonitoring studijama zbog svojih karakteristika: slabe pokretljivosti, načina ishrane, brojnosti, veličine tela i dr. Oni imaju sposobnost da u svom telu akumuliraju znatne količine zagađujućih materija. Na osnovu analize sadržaja ovih materija u mekušcima moguće je zaključivati o kvalitetu sredine. Cilj ove doktorske disertacije bio je da se ispita uticaj različitih lokaliteta: reka Dunav, Tisa i Velika Morava na promene u parametrima antioksidacionog zaštitnog sistema: superoksid-dismutaze (SOD, EC 1.15.1.1), katalaze (CAT, EC 1.11.1.6), glutationperoksidaze (GSH-Px, EC 1.11.1.9), glutation-reduktaze (GR, EC 1.6.4.2), enzima faze II biotransformacije glutation-S-transferaze (GST, EC 2.5.1.18), kao i koncentraciju glutationa (GSH). Parametri su ispitivani u visceralnoj masi puža Viviparus acerosus i školjke Corbicula fluminea. Interspecijske razlike su praćene analizom parametara antioksidacionog zaštitnog sistema u visceralnoj masi puževa V. acerosus, Amphimelania holandrii i školjki C. fluminea i Sinanodonta woodiana iz Velike Morave. Uticaj sezonskih faktora određivan je tokom aprila i septembra u visceralnoj masi vrste V. acerosus iz Dunava, Tise i Velike Morave. Analizirane su koncentracije 15 metala u visceralnoj masi sve četiri vrste slatkovodnih mekušaca u aprilu. Značajne razlike aktivnosti SOD u visceralnoj masi puža V. acerosus iz različitih reka čine ovaj enzim pogodnim biomarkerom u ekofiziološkim studijama. Varijacije aktivnosti GST u visceralnoj masi školjke C. fluminea iz različitih reka, ukazuju na to da se i ovaj enzim može koristiti kao biomarker. Uporedni pregled parametara antioksidacionog zaštitnog sistema četiri vrste slatkovodnih mekušaca izdvajaju A. holandrii kao potencijalnu bioindikatorsku vrstu. Odsustvo jasnog sezonskog uticaja na GST u visceralnoj masi V. acerosus izdvaja ovaj enzim kao potencijalno dobar biomarker...Freshwater molluscs are a good model organism for studying the effects of environmental factors in biomonitoring studies due to their following characteristics: poor mobility, diet, numbers, size. They have the ability to accumulate in their body significant amounts of pollutants. By analysing the contents of these substances in molluscs we can draw conclusions about the quality of the environment. The aim of this PhD thesis was to investigate the effects of different localities, the Danube, Tisa and Velika Morava Rivers, on changes in the parameters of the antioxidative defence system: the enzymatic activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), glutathione-peroxidase (GSH-Px; EC 1.11.1.9), glutathionereductase (GR; EC 1.6.4.2), and phase II biotransformation enzyme glutathione-S-transferase (GST; EC 2.5.1.18), and glutathione (GSH) concentration. The parameters were investigated in the visceral mass of the snail Viviparus acerosus and mussel Corbicula fluminea. Interspecies differences were followed by analysing the parameters of the antioxidative defense system in the visceral mass of snails, V. acerosus, Amphimelania holandrii, and mussels, C. fluminea and Sinanodonta woodiana, from the Velika Morava River. Seasonal changes were determined during April and September in the visceral mass of species, V. acerosus from the Danube, Tisa and Velika Morava. We analyzed the concentrations of 15 metals in the visceral mass of all four species of freshwater molluscs in April. Significant differences in SOD activities in the visceral mass of the snail V. acerosus from different rivers render this enzyme a suitable biomarker for ecophysiological studies. Variations in GST activities in the visceral mass of the mussel C. fluminea from different rivers can also be considered as a good biomarker. A comparative review of the parameters of the antioxidative defence system in four species of freshwater molluscs distinguish A. holandrii as a potential bioindicator species. The absence of a clear seasonal impact on GST in the visceral mass of V. acerosus makes this enzyme a good biomarker. The potential of freshwater molluscs to bioaccumulate metals studied in this dissertation showed that V. acerosus is a good indicator of metal pollution in river systems..

Glutathione as a suitable biomarker in hepatopancreas, gills and muscle of three freshwater crayfish species

We determined the contents of total glutathione (tGSH), reduced glutathione (GSH), and oxidized glutathione (GSSG) and values of the glutathione redox index (GSH RI) in hepatopancreas, gills, and muscle of three freshwater crayfish species: noble crayfish (Astacus astacus) from the Southern Morava River, stone crayfish (Austropotamobius torrentium) from the Krajkovačka River, and spinycheek crayfish (Orconectes limosus) from the Danube River. The obtained data show strong tissue and species specifity of investigated parameters: tGSH, GSH, GSSG, and GSH RI in the hepatopancreas, gills, and muscle of the indicated crayfish species. Our work represents the first study of its kind and showed that the investigated parameters can be considered suitable biomarkers of the cellular glutathione redox status in of freshwater crayfish species.Određivan je sadržaj ukupnog glutationa (tGSH),redukovanog glutationa (GSH),oksidovanog glutationa (GSSG)i glutation redoks indeksa (GSH RI)u hepatopankreasu, škrgama i mišićima tri vrste slatkovodnih rakova: rečnog raka (Astacus astacus) iz reke Južna Morava, raka kamenjara (Austropotamobius torrentium) iz Krajkovačke reke i američkog raka (Orconecteslimosus) iz reke Dunav. Dobijeni podaci pokazuju značajnu tkivnu i specijes specifičnost ispitivanih parametara: GSH, GSSG i GSHRI u hepatopankreasu, škrgama i mišićima kod rakova. Naš rad predstavlja prvu studiju ove vrste i pokazuje da ispitivani parametri mogu biti pogodni biomarkeri ćelijskog redoks statusa glutationa kod slatkovodnih vrsta rakova.Projekat ministarstva br. 143035

Seasonal changes in oxidative stress biomarkers of the snail Viviparus acerosus from the Velika Morava River, Serbia

The river snail (Viviparus acerosus) from the Velika Morava River, Serbia was chosen in our study in order to determine seasonal changes in oxidative stress biomarkers between July (summer) and September (autumn). The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST), as well as the concentration of total glutathione (GSH), were examined in the whole body of the river snails. The obtained results show significantly higher activities of CAT, GSH-Px, GR and biotransformation phase II enzyme GST in September compared to July, while the GSH concentration was lower. There was no general trend in the seasonal changes in the activity of SOD. The presented data show that animals in their natural environment are exposed to constant fluctuations of environmental conditions that could cause antioxidants to exhibit seasonal variations. This fact should be considered as an important variable in the interpretation of results in biomonitoring studies.Projekat ministarstva br. 173041 i br. 17302

Antioxidant Response in Gills of Eurasian Perch ( Perca fluviatilis ) to Cyanobacterial Bloom Exposure in the Gruža Reservoir

The aim of this study was to assess the impact of an Aphanizomenon flos-aquae bloom in the Gruža Reservoir on the antioxidant parameters measured in the gill cells of Eurasian perch (Perca fluviatilis). Effects of the bloom were evaluated through copper, zinc and manganese containing superoxide dismutase (CuZnSOD and MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and biotransformation phase II enzyme glutathione-S-transferase (GST) activities and glutathione (GSH) and sulfydryl (–SH) groups concentrations. The activities of CuZn SOD and Mn SOD decreased during the cyanobacterial bloom compared to the period before the bloom. The opposite trend was observed for CAT and GSH-Px activities that increased. During the bloom GR and GST activities and GSH concentration decreased significantly. CuZn SOD, GR and GST activities are detected as the most important parameters for individual variations. Our results indicated that an A. flos-aquae bloom promotes oxidative stress in the gills of P. fluviatilis. The investigated parameters could be used in environmental risk assessment procedures to monitor the effects of A. flos-aquae blooms on fish. Observed changes in the antioxidant parameters in P. fluviatilis gills make them potential biomarkers in the research of ecological risk situations in freshwater ecosystems with frequent cyanobacterial blooms.Water Research and Management (2016), 6(2): 19-2

Activity of oxidative stress biomarkers in the white muscle of red mullet (Mullus barbatus L.) from the Adriatic sea

The aim of this study was to investigate the activity of oxidative stress biomarkers (total superoxide dismutase - Tot SOD; and copper and zinc-containing superoxide dismutase - CuZn SOD; manganese-containing superoxide dismutase - Mn SOD; catalase - CAT; glutathione peroxidase - GSH-Px; and glutathione reductase - GR), as well as the biotransformation phase II enzyme glutathione-S-transferase (GST), in the white muscle of red mullet (Mullus barbatus L.) at Platamuni (PL) and Valdanos (VAL) in the Adriatic Sea during the winter and spring seasons. The obtained results show both site and seasonal influences on the investigated parameters, with lower enzyme activities at VAL than at PL and in spring than in winter.Cilj ovog rada bio je ispitivanje aktivnosti biomarkera oksidacionog stresa(ukupne superoksid-dismutaze-Tot SOD, bakar cink sadržavajuće superoksid-dismutaze-CuZn SOD, mangan sadržavajuće superoksid-dismutaze-Mn SOD, katalaze-CAT, glutation-peroksidaze-GSH-Px i glutation-reduktaze-GR) kao i enzimafaze II biotransformacije glutation-S-transferaze (GST) u belom mišiću trlje (Mullus barbatus L.) sa lokaliteta Platamuni i Valdanosu Jadranskom moru (Crna Gora) u zimskoj i prolećnoj sezoni. Dobijeni rezultati pokazuju razlike ispitivanih parametara između različitih lokaliteta kao i ispitivanih sezona sa nižim aktivnostima enzima u VAL nego u PL i u proleće u odnosu nazimu.Projekat ministarstva br. 143035

The Effect of Shelter on Oxidative Stress and Aggressive Behavior in Crested Newt Larvae (Triturus spp.)

Shelters are important for animal survival. Provision of adequate hiding places allow animals to express their natural sheltering behavior and it can have different positive effects on cortisol levels, physiological processes and mental performance. Although the absence of a refuge activates some stress response, its effect on oxidative stress has not been adequately examined. This study investigated whether the presence/absence of a shelter modifies the oxidative status (the antioxidant system and oxidative damage) and aggressive behavior of crested newt larvae (Triturus macedonicus and its hybrid with T. ivanbureschi). Our results show that individuals reared with shelters had lower values of the tested antioxidant parameters (catalase, glutathione peroxidase, glutathione S-transferase and glutathione), indicating a lower production of reactive species than individuals reared without shelter. The same pattern was observed in both T. macedonicus and its hybrid. Contrary to the activation of some physiological pathways, shelter availability did not significantly affect the rate of intraspecific aggressive behavior. The physiological benefits of shelter use can be manifested as a lower requirement for investment in the energy necessary for the maintenance of the upregulated antioxidant defenses, activation of repair systems and synthesis of endogenous antioxidants. This study highlights the importance of shelter provision, which may be valuable in habitat restoration and animal conservation studies

Superoxide dismutase and catalase activities in the digestive gland and gills of the freshwater bivalve Unio pictorum from the Sava river

We investigated the potential use of the antioxidant defense enzymes in freshwater mussel (Unio pictorum) as biomarkers of oxidative stress. The enzymatic activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), total protein concentration in addition to protein and SOD electrophoretic profiles were examined in the digestive gland and gills of the freshwater bivalve Unio pictorum at two localities on the River Sava. The differences between SOD and CAT activities in examined tissues of freshwater bivalve Unio pictorum reflect dissimilar metabolic and antioxidative activities and this can be the result of both tissue or locality specificities and diverse ecophysiological influences on the organism.Projekat ministarstva br. 173041

Concentration of antioxidant compounds and lipid peroxidation in the liver and white muscle of hake (Merluccius merluccius L.) in the Adriatic sea

Specimens of a marine freshwater fish - hake (Merluccius merluccius L.) - were collected at the localities of Platamuni and Valdanos (Montenegro, South Adriatic) in the spring of 2003. Our results showed higher concentrations of LPO, Vit C, and Vit E in the liver in comparison with white muscle. The concentration of LPOin both tissues was higher, while that of Vit E was lower at Valdanos compared to Platamuni. These differences in parameters of oxidative stress are partly due to differences in temperature and the concentrations of nitrites, nitrates, and detergents in the waters of Valdanos compared to Platamuni.Tokom 2003. godine uzorkovane su jedinke morske ribe - oslića (Merluccius merluccius L.) iz voda sa lokaliteta Platamuni i Valdanos (Crna Gora, južni Jadran) u prolećnom periodu. Dobijeni rezultati su pokazali veće koncentracije LP, Vit Ci Vit E u jetri oslića u odnosu na belo mišićno tkivo. Koncentracija LP je bila veća, a Vit E manja u oba ispitivana tkiva oslića u vodama lokaliteta Valdanos u poređenju sa Platamuni. Ovakve promene parametara oksidacionog stresa posledica su promena temperature, kao i povećanih koncentracija nitrita, nitrata i deterdženata u vodama lokaliteta Valdanos u odnosu na Platamune.Projekat ministarstva br. 143035

Heavy metals, oxidative stress and their relationship in Pelophylax ridibundus frogs during pre- and posthibernation period

In temperate regions, low temperatures and the lack of food during the winter lead the water frogs to hibernate in order to survive. Hibernation represents an adaptation mechanism and is a part of frogs yearly life cycle (Wells, 2007). Hibernation, as well as pre- (autumn) and post-hibernation (spring) periods are characterized by different biochemical, physiological, morphological and behavioral changes, all in order to prepare the organisms for a significant depression in aerobic metabolic rate (autumn) and then recovery to a normal state (spring). An important role in this response plays the antioxidative defense system (AOS), especially in spring, when increased metabolic rate during arousal is followed by high oxygen consumption and reactive oxygen species (ROS) generation. Oxidative stress is also induced by various xenobiotics in the environment and this raises the question of their share in oxidative stress of hibernators in a yearly cycle? To examine this we first determined concentrations and seasonal variations of accumulated metals (As, Cd, Cr, Fe, Hg, Ni, Pb, Zn) and AOS parameters (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH- Px), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) and sulfhydryl (SH) groups concentrations) in skin and muscle of Pelophylax ridibundus during pre- (autumn) and post- hibernation (spring) periods, and second we evaluated the possible relationship between accumulated metals and AOS parameters. The results showed seasonal variation of accumulated concentrations: autumn frogs were characterized by higher concentrations of Ni, Cr and Hg, while frogs sampled in spring had higher As and Cd. On the other hand almost all AOS parameters were higher during the spring in comparison to individuals from autumn (in skin SOD, CAT, GR, GST, GSH and SH groups, while in muscle SOD, CAT, GSH-Px, GR and SH groups). The number of significant correlations between accumulated metals and AOS parameters were very similar in frogs from both seasons. All results showed that oxidative stress in the post-hibernating season was mainly caused by the organism’s recovery from hibernation, and that the metals did not contribute significantly to this response, indicating that natural selection affected AOS of the frogs in that way that during arousal they were able to produce higher levels of antioxidant defenses

Heavy metals, oxidative stress and their relationship in Pelophylax ridibundus frogs during pre- and posthibernation period

In temperate regions, low temperatures and the lack of food during the winter lead the water frogs to hibernate in order to survive. Hibernation represents an adaptation mechanism and is a part of frogs yearly life cycle (Wells, 2007). Hibernation, as well as pre- (autumn) and post-hibernation (spring) periods are characterized by different biochemical, physiological, morphological and behavioral changes, all in order to prepare the organisms for a significant depression in aerobic metabolic rate (autumn) and then recovery to a normal state (spring). An important role in this response plays the antioxidative defense system (AOS), especially in spring, when increased metabolic rate during arousal is followed by high oxygen consumption and reactive oxygen species (ROS) generation. Oxidative stress is also induced by various xenobiotics in the environment and this raises the question of their share in oxidative stress of hibernators in a yearly cycle? To examine this we first determined concentrations and seasonal variations of accumulated metals (As, Cd, Cr, Fe, Hg, Ni, Pb, Zn) and AOS parameters (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH- Px), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) and sulfhydryl (SH) groups concentrations) in skin and muscle of Pelophylax ridibundus during pre- (autumn) and post- hibernation (spring) periods, and second we evaluated the possible relationship between accumulated metals and AOS parameters. The results showed seasonal variation of accumulated concentrations: autumn frogs were characterized by higher concentrations of Ni, Cr and Hg, while frogs sampled in spring had higher As and Cd. On the other hand almost all AOS parameters were higher during the spring in comparison to individuals from autumn (in skin SOD, CAT, GR, GST, GSH and SH groups, while in muscle SOD, CAT, GSH-Px, GR and SH groups). The number of significant correlations between accumulated metals and AOS parameters were very similar in frogs from both seasons. All results showed that oxidative stress in the post-hibernating season was mainly caused by the organism’s recovery from hibernation, and that the metals did not contribute significantly to this response, indicating that natural selection affected AOS of the frogs in that way that during arousal they were able to produce higher levels of antioxidant defenses