Branka R. Gavrilović

Comparative study of oxidative stress parameters and acetylcholinesterase activity in the liver of Pelophylax esculentus complex frogs

Comparative activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST), the concentrations of total glutathione (GSH), sulfhydryl groups (–SH) and the activity of the neurotoxicity biomarker acetylcholinesterase (AChE) were investigated in the livers of species belonging to the Pelophylax esculentus “complex” (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax kl. esculentus) from the wetland, Obedska bara in Serbia. The condition factor (CF) and hepato somatic index (HSI) were also calculated. All three species were caught at same locality and were exposed to the same environmental conditions. Liver SOD activity was lower in P. ridibundus than in P. kl. esculentus and P. lessonae; higher activities of CAT, GR and GST were observed in P. kl. esculentus frogs as compared to their parental species. The activity of GSH-Px was significantly lower in P. kl. esculentus. The activity of AChE was increased in P. lessonae as compared to P. kl. esculentus and P. ridibundus. Similar concentrations of GSH and —SH groups were observed in all investigated species. P. kl. esculentus had a higher CF, while the HSI was lower when compared to the parental species. Our findings suggest that the parental species (P. ridibundus and P. lessonae) possess more similar antioxidative responses to environmental conditions than the hybrid species P. kl. esculentus. The obtained results improve our understanding of the biology and physiology of these three closely related species.

Biomarkers of oxidative stress and metal accumulation in marsh frog (Pelophylax ridibundus)

To understand the effect of metals on the marsh frog Pelophylax ridibundus and the possible environment-induced changes in oxidative stress enzymes, we determined the concentrations of 18 metals: Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Ga, Hg, In, Li, Mn, Ni, Pb, Sr, and Zn, in the tissues (liver, skin, and muscle) and water samples collected from different locations in Serbia. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), acetylcholinesterase (AChE), and changes in concentrations of reduced glutathione (GSH) and sulfhydryl groups (SH) were analyzed in the tissues of the sampled frogs. The concentrations of Al, Cd, Co, Cr, Cu, Fe, Ga, Hg, and Ni were highest in the liver, whereas those of Ba, Ca, Li, Mn, Pb, Sr, and Zn were highest in the skin. Hg correlated positively with liver SOD (in frogs from Danube-Tisza-Danube Canal (DTD)), muscle CAT (DTD), and muscle GST Ponjavica River (PO); Pb demonstrated a strong positive correlation with liver GR in frogs from Mt. Fruška Gora (FG); Cd only exhibited a positive correlation with AChE in the skin of frogs from DTD. In the skin, Zn correlated positively with AChE (DTD), SH groups (PO), and CAT (FG), and negatively with CAT, GST, and SH in the liver of frogs from DTD. Examination of these oxidative stress biomarkers, together with analysis of metal accumulation in the liver and skin of marsh frogs, provides a powerful tool for the assessment of metal pollution.

Antioxidative responses of the tissues of two wild populations of Pelophylax kl. esculentus frogs to heavy metal pollution

Heavy metal pollution of the aquatic environment is of great concern worldwide. Heavy metals are capable of inducing oxidative stress by increasing the formation of reactive oxygen species (ROS), and directly affecting the antioxidant defense system (AOS) in living organisms. The frog Pelophylax kl. esculentus is a semiaquatic species with semipermeable skin and a complex lifecycle, and represents a potentially useful bioindicator organism. The aim of this study was to investigate the accumulation of several heavy metals (Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb and Zn), and their effects on selected parameters of the AOS, including the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), phase II biotransformation enzyme glutathione-S-transferase (GST), the total glutathione (GSH) contents and sulfhydryl (SH) group concentrations, as well as cholinesterases (ChEs) activities in the liver, skin and muscle of P. kl. esculentus. Frog samples were collected at two sites (the Danube-Tisza-Danube canal (DTDC) and the river Ponjavica) in Serbia, which are characterized by different levels of metal pollution. Differences between the metal contents in different tissues showed that the skin of frogs from the DTDC accumulated statistically higher concentrations of Cd, Cu, Pb and Zn, while only the Fe concentration was lower. No significant differences between metal concentrations in muscle tissues of frogs from the DTDC and Ponjavica were observed. Examination of the parameters of the AOS revealed that frogs from the DTDC had higher concentrations of GSH in the liver and of SH groups in the skin and muscle, whereas the activities of the antioxidative enzymes SOD, GHS-Px and GR in the liver and of GR in the skin were lower than in frogs from the Ponjavica. The relationship between metal concentrations and AOS parameters showed the highest number of correlations with GSH, GR and CAT, and with Ni, Zn, Hg, Cr and Cd. Based on the results in this study, we concluded that increased concentrations of heavy metals in frog tissues can alter the AOS, which leads to higher concentrations of GSH and SH groups and lower activities of antioxidative enzymes. The response of the AOS to metal pollutants allowed us to make a distinction between different frog tissues, and to conclude that the liver and skin are more suitable for assessing metal-induced oxidative stress in frogs than muscle.

Biomarkers of oxidative stress and acetylcholinesterase activity in the blood of grass snake (Natrix natrix L.) during prehibernation and posthibernation periods

This work examined the enzymatic (superoxide dismutase-CuZn SOD, catalase-CAT, glutathione peroxidase-GSHPx, glutathione reductase-GR, and the biotransformation phase II enzyme glutathione-S-transferase-GST) and nonenzymatic (total glutathione-GSH and lipid peroxides-TBARS concentrations) biomarkers of oxidative stress and acetylcholinesterase (AChE) activity in the blood of the grass snake (Natrix natrix L.) during prehibernation and posthibernation. The animals were collected in October (prehibernation) and April (posthibernation) at the nature reserve Obedska Bara (OB) and industrial region Pancevacki Rit (PR) in Serbia. In posthibernation, decreased CAT activity and TBARS concentration in specimens from PR, and decreased GR and AChE activities, and TBARS concentration in specimens from OB were observed, whereas GR and GST activities and GSH concentration were significantly elevated in the specimens from PR. In prehibernation, CAT activity and GSH concentration were increased, while GSH-Px, GR, GST and AChE activities and TBARS concentration were decreased in the specimens from PR when compared to animals from OB. During the posthibernation, the activity of CuZn SOD was decreased, while GST and AChE activities were increased in the specimens from PR when compared to the specimens from OB. These differences represented an adaptive mechanism to oxidative stress induced by tissue reoxygenation during arousal from hibernation and could be modulated by environmental pollution.

Oxidative stress biomarkers, cholinesterase activity and biotransformation enzymes in the liver of dice snake (Natrix tessellata Laurenti) during pre-hibernation and post-hibernation: A possible correlation with heavy metals in the environment

We investigated in the liver of dice snakes during pre- and post-hibernation changes in the following antioxidant parameters: total, manganese and copper zinc containing superoxide dismutases (Tot SOD, MnSOD, CuZn SOD, respectively), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the concentrations of total glutathione (GSH) and sulfhydryl groups (-SH). In addition, we examined the expression of phase I biotransformation enzyme cytochrome P4501A (CYP1A) and the activity of phase II biotransformation enzyme glutathioneS-transferase (GST), the level of lipid peroxidation (by measuring the thiobarbituric acid-reactive substances (TBARS)), cholinesterase activity (ChE) and metallothionein expression (MT). We also measured the concentrations of heavy metals, including Al, Cd, As, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb and Zn in the water and snake liver during both periods. During the post-hibernation period, the activities of Tot SOD, CuZn SOD and GST and the concentration of GSH were significantly decreased, while GSH-Px and GR activities, the concentrations of -SH groups and TBARS were significantly increased. The activities of Mn SOD, CAT and ChE, and the relative amounts of CYP1A and MT did not significantly change during the investigated periods. The observed differences in the examined parameters probably represent adaptive physiological responses to sudden changes in tissue oxygenation during arousal from hibernation. Our findings also indicate that the accumulated metals modulated the responses of the examined parameters during the investigated periods.

Sublethal effects of the pyrethroid insecticide deltamethrin on oxidative stress parameters in green toad (Bufotes viridis L.)

Pyrethroids are synthetic insecticides widely used in agriculture, public health, and veterinary medicine. Deltamethrin, a type II pyrethroid insecticide, has attracted particular attention because of its frequent use. The mechanisms of the toxicity of most pesticides (including pyrethroids) in nontarget organisms is linked to the production of free radicals, oxidative stress induction, increased lipid peroxidation, and disruption of the total antioxidant potential. The aim of the present study was to investigate the effects of acute toxicity of 3 different concentrations (8, 16, and 32 mg/kg body wt) of orally applied deltamethrin after 96 h of treatment. Some of the front-line oxidative stress parameters as well as cholinesterase (ChE) activity and cytochrome P4501A (CYP1A) expression in the liver, muscle, skin, and gastrointestinal tissue of the adult green toad Bufotes viridis were determined. Compared with the control group, the activity of catalase and glutathione reductase was increased in the liver and skin, while the concentration of sulfhydryl groups was reduced. In the liver and muscle, concentrations of thiobarbituric reactive substances were increased, as well as liver CYP1A expression. In the muscle and skin, glutathione-S-transferase activity was higher in treated toads. The oxidative stress parameters examined were affected by different deltamethrin concentrations. We conclude that the assessed parameters represent good biomarkers of pesticide-induced oxidative stress. Environ Toxicol Chem 2017;36:2814-2822.

Bioaccumulation and effects of metals on oxidative stress and neurotoxicity parameters in the frogs from the Pelophylax esculentus complex

Metals are involved in the formation of reactive oxygen species and can induce oxidative stress. The aim of this study was to assess the effects of several metals on oxidative stress in the skin and muscle of the Pelophylax esculentus “complex” frogs (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax esculentus) that inhabit the wetland Obedska Bara in Serbia, and the potential use of these species as bioindicator organisms in biomonitoring studies. The biomarkers of oxidative stress (SOD, CAT, GSH-Px, GR, GST activities and GSH, SH concentrations) and cholinesterase activity were investigated. The concentrations of nine metals (Fe, Cu, Zn, As, Cd, Cr, Hg, Ni, and Pb) were measured in the water and tissues. Correlations were established between metals and biomarkers in the tissues. The results of metal accumulation distinguished the skin of P. lessonae and muscle of P. ridibundus from other P. esculentus complex species. The oxidative stress biomarkers observed in P. ridibundus and P. esculentus had greater similarity than in P. lessonae. The P. lessonae displayed the highest number of correlations between biomarkers and metals. The results of tissue responses revealed that skin was more susceptible to metal-induced oxidative stress, with only exception of As. In the light of these findings, we can suggest the use of P. esculentus complex species as a biomonitoring species in studies of metal accumulation and metal-induced oxidative stress, but with special emphasis on P. lessonae.

Comparative assessment of the antioxidative defense system in subadult and adult anurans: A lesson from the Bufotes viridis toad

During their complex life cycle, anurans are faced with various physiological and biochemical demands that can result in increased free radical production. The antioxidative system (AOS), assumes a central role in protection from oxidative stress, and increased knowledge of its response would allow us to identify and quantify underlying costs of free radical production. In this study, we compared the patterns and levels of integration of the AOS during two life stages of Bufotes viridis toads from natural populations: young, fully developed, reproductively inactive subadults and reproductively active adults. All examined parameters (SOD, CAT, GSH-Px, GR, GST, GSH and SH groups) were measured in the liver, skin and muscle. The obtained results showed that: (1) subadult individuals possessed a more integrated AOS (a higher index of integration and number of significant correlations), based mostly on the coordinated action of SOD-CAT; (2) adult individuals used the GSH-Px/GSH system; (3) all examined tissues exhibited similar patterns of variation of AOS parameters; (4) significant correlations between the AOS and biometric parameters. Our results indicate that adult and subadult toads differed in the way they processed free radicals, implying that adults may be more susceptible to oxidative damage. The knowledge from this study will contribute to a better understanding of the AOS and how its activity is modulated in natural populations of anurans throughout their life.

Oxidative cost of interspecific hybridization: a case study of two Triturus species and their hybrids.

ABSTRACT Oxidative stress has most recently been suggested as one of the possible mechanisms responsible for reduced fitness of hybrids. To explore possible oxidative cost of hybridization, we examined anti-oxidant defence system parameters (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glutathione, SH groups), their interconnectedness (index of integration) and levels of oxidative damage [concentrations of lipid peroxides, TBARS (thiobarbituric acid reactive substances)] in laboratory-reared newt species, Triturus macedonicus and T. ivanbureschi, and their hybrid. Our results showed that parental species differed in anti-oxidant defence system parameters, but not in the levels of integration of the whole system and oxidative damage. Individuals of T. ivanbureschi had higher activities of superoxide dismutase, glutathione S-transferase and concentrations of glutathione. Hybrid individuals of crested newts displayed higher levels of the anti-oxidant defence system (higher superoxide dismutase, catalase, glutathione peroxidase activities and concentrations of SH groups), and a lower overall correlation of anti-oxidant system (lower index of integration) in comparison with both parental species, suggesting that they may possess a less efficient anti-oxidant defence system and a higher investment in maintaining oxidative balance. The higher investment in the anti-oxidant system could divert limited resources away from other functions and affect further hybrid fitness. The presented findings contribute to a better understanding of the anti-oxidant defence system of crested newts and their interspecies differences, and support the hypothesis that oxidative stress is one of the costs of interspecific hybridization. Summary: Hybrid individuals of crested newts had higher values of anti-oxidant parameters and a less integrated anti-oxidant defence system in comparison with parental species, indicating a higher investment in maintaining oxidative balance.

Antioxidant parameters in fish white muscle as biomarkers of exposure to a cyanobacterial bloom

Abstract This study examines the responses of antioxidant parameters in the white muscle of four freshwater fish species: Rutilus rutilus (roach), Blicca bjoerkna (white bream), Carassius gibelio (Prussian carp) and Perca fluviatilis (European perch) after exposure to a bloom of Aphanizomenon flos-aquae (Cyanobacteria) in the Gruža Reservoir in Serbia. The set of analyzed biomarkers included the activities of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and biotransformation phase II enzyme glutathione-S-transferase (GST), and the concentrations of total glutathione (GSH) and sulfhydryl (-SH) groups that were measured before and during the bloom. Our results show that SOD activity in C. gibelio and P. fluviatilis, GSH-Px and GST activities in all four fish, GR activity in R. rutilus and C. gibelio and the -SH group concentration in P. fluviatilis markedly decreased during the bloom. The activity of CAT in all four fish and the concentration of GSH in B. bjoerkna and P. fluviatilis significantly increased. The observed changes in nonspecific antioxidant biomarkers in fish muscle (especially CAT, GSH-Px and GST activities) suggest that these parameters could be useful tools in environmental monitoring programs of the freshwater ecosystems where cyanobacterial blooms occur.