Responses of the Antioxidant and Osmoregulation Systems of Fish Erythrocyte Following Copper Exposures in Differing Calcium Levels.

Freshwater fish Oreochromis niloticus were exposed to Cu in differing Ca2+ levels (15, 30 and 90 mg/L), using acute (0.3 µM, 3 d) and chronic (0.03 µM, 30 d) exposure protocols and enzyme activities related to the antioxidant (catalase, CAT, EC 1.11.1.6; superoxide dismutase, SOD, EC 1.15.1.1; glutathione peroxidase, GPx, EC 1.11.1.9) and osmoregulation (Total, Na+/K+-ATPase, EC 3.6.3.9, Mg2+-ATPase, EC 3.6.3.2) systems in the erythrocytes were measured. Activities of antioxidant enzymes generally decreased significantly following either Ca2+ alone or Ca2++Cu combinations in both acute and chronic exposures. Na+/K+-ATPase activity significantly decreased in chronic exposures, though there was no clear trend in acute exposures. Mg2+-ATPase activity increased significantly in acute exposures, but not in chronic ones. There were more significant alterations in acute exposure compared to chronic ones. There was no clear trend regarding Cu toxicity and its relationship with Ca2+, which may possibly be prompted by the compensatory mechanisms of the enzymes. It may be concluded that freshwater fish erythrocytes may face different degrees of more physiological stress from different waters.

Effects of heavy metals (Cd, Cu, Cr, Pb, Zn) on fish glutathione metabolism.

The glutathione metabolism contains crucial antioxidant molecules to defend the organisms against oxidants. Thus, the aim of this study was to investigate the response of the glutathione metabolism in the liver of freshwater fish Oreochromis niloticus exposed to metals (Cu, Cd, Cr, Pb, Zn) in different periods. Fish were exposed to metals (as 1 µg/mL) individually for 1, 7, and 14 days and subsequently antioxidant enzymes (glutathione peroxidase, GPX; glutathione reductase, GR and glutathione S-transferase, GST) and glutathione levels (total glutathione, tGSH; reduced glutathione, rGSH; oxidized glutathione, GSSG and GSH/GSSG ratios) in the liver were measured. There was no fish mortality during the experiments, except Cu exposure. The antioxidant enzymes responded differently to metal exposures depending on metal types and exposure durations. GPX activity increased only after Cd exposure, while GST activity increased following 7 days of all metal exposures. However, GR activity did not alter in most cases. Total GSH and GSH/GSSG levels generally decreased, especially after 7 days. Data showed that metal exposures significantly altered the response of antioxidant system parameters, particularly at day 7 and some recovery occurred after 14 days. This study suggests that the response of antioxidant system could help to predict metal toxicity in the aquatic environments and be useful as an "early warning tool" in natural monitoring studies.

Effects of fish size on the response of antioxidant systems of Oreochromis niloticus following metal exposures.

The size of a fish is an important factor in its physiology, and metal uptake is affected by animal physiology. In this study, small and large tilapias (Oreochromis niloticus) differing approximately twofold in length and fivefold in weight were compared for their antioxidant response. Both groups were exposed to Cu or Cr (1.0 µg/mL) in a freshwater (-80 mg CaCO3/L, conductivity 1.77 mS/cm) using 2 exposure protocols (20 µM for 48 h and 10 µM for 6 days). Following the exposures, the antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; glutathione reductase, GR and glutathione S-transferase, GST) and glutathione (GSH) levels were measured in the liver of fish. Results showed that small fish was affected from exposure conditions much more than large ones as their antioxidant parameters significantly decreased even in controls. Metal exposures of small fish caused significant increases in SOD and CAT activity in acute Cu or Cr exposures. Subchronic Cr exposure of small fish also caused significant increases in CAT, GPx and GST activities, while there was no significant change in Cu-exposed ones. Large fish, however, showed different antioxidant responses as their levels mostly decreased. This study demonstrated that the response of antioxidant system in the liver of tilapia varied in relation to fish sizes and emphasized using different size groups in environmental monitoring and also in evaluation of fish biomarkers.

The effects of increased freshwater salinity in the biodisponibility of metals (Cr, Pb) and effects on antioxidant systems of Oreochromis niloticus.

Anthropogenic activities can increase the salinity of freshwaters and this may cause stress for fish and affect metal bioavailability. Oxidative stress biomarkers are of great interest due to their responses to environmental stressors which provide valuable data for biological monitoring of aquatic pollution. Thus, the individual and combined effects of salinity and metals (Cr, Pb) were investigated in the liver of freshwater fish Oreochromis niloticus in the present study. Fish were exposed to salinity (2 and 8 ppt) alone and salinity+metal (1 µg/mL Pb and Cr) combination exposures for 0, 1, 7 and 14 days and subsequently antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPX; glutathione reductase, GR and glutathione S-transferase, GST) activities and glutathione (GSH) levels in the liver were measured. Data showed that all the parameters varied in relation to metal species, exposure durations and salinity levels. Profound alterations on the measured parameters were detected at the lower salinity compared to the higher one. Salinity increase effectively stimulated the antioxidant parameters. The effects of salinity and metals on the measured parameters increased as the exposure duration prolonged. SOD was the most affected antioxidant parameter from both salinity and metals. Because metal and salinity stresses affect fish antioxidant system, this work suggests that the chemistry of freshwaters should be taken into account in natural monitoring for metal contamination in the field.

Natural occurrence of metallothioneinlike proteins in liver tissues of four fish species from the northeast Mediterranean Sea.

Fish with different ecological needs were captured from the northeast Mediterranean Sea to determine metallothioneinlike proteins in their livers. Heat-treated liver samples were run on a column packed with Sephadex G-75 (Sigma, Germany) for subsequent determination of metals, sulfhydryl, and absorbance at 254 and 280 nm. Liver cytosols of Sparus auratus and Mullus barbatus had three heat-stable protein peaks, whereas Mugil cephalus and Atherina hepsetus had two. The third protein peak, containing metallothionein-like proteins (5000 to 6000 Da), was present in all fish. Only high- and low-molecular-weight proteins contained sulfhydryl and metals. Zinc was the most abundant metal in the metallothioneinlike proteins, whereas cadmium and lead were not detected. The differences in the characteristics of metal-binding proteins among fish indicate that the biological characteristics of fish species should be carefully taken into account concerning natural monitoring studies.