Oxidative stress in Pelophylax esculentus complex frogs in the wild during transition from aquatic to terrestrial life.

During life, anuran individuals undergo drastic changes in the course of transition from aquatic to terrestrial habitat, when they are faced with metabolically demanding processes (growth, responses to developmental pressures), which result in increased production of reactive oxygen species (ROS), signaling molecules involved in development that can induce oxidative damage and stress. This situation can be further complicated by environmental influences. The aim of this study was to investigate oxidative stress parameters in naturally developing Pelophylax esculentus complex frogs during four developmental periods: premetamorphosis, prometamorphosis, metamorphic climax and juvenile stage, in order to examine changes in the response of the antioxidative system (AOS) and oxidative damage during the transition from aquatic to terrestrial life. Results show that ontogenetic shifts in anurans are accompanied by different levels of damage and AOS responses, which vary from the increased first-line enzymatic activities during the early period of development (premetamorphosis), through increased changes in the non-enzymatic complement during the metamorphic climax, to changes in both the enzymatic and non-enzymatic components observed in juvenile individuals. Premetamorphic individuals and individuals in metamorphosis displayed higher levels of lipid peroxidation, indicating that direct exposure to the environment for the first time and the modulation of organs are the most susceptible stages for oxidative damage. On the other hand, lower oxidative damage in juveniles points to the ability of their AOS to efficiently respond to challenges of the terrestrial environment. This study highlights the importance of ROS and the AOS of anurans in response to different developmental and/or environmental pressures that individuals face.

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.

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.

Age-related changes in antioxidant defenses of the Mediterranean centipede Scolopendra cingulata (Chilopoda).

The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST), as well as the concentrations of sulfhydryl (SH) groups and glutathione (GSH) were analyzed in five age classes of the Mediterranean centipede Scolopendra cingulata as follows: embryo, adolescens, maturus junior, maturus, and maturus senior. The data obtained showed the presence of SOD, CAT, GSH-Px, GR, GST, and SH groups in embryos. The transition from embryo to adolescens was accompanied by an increase in the activities of all studied enzymes, in response to the increased production of ROS due to the increased metabolic activity of the centipede associated with growth and development. Our results show that trends in antioxidant enzyme (AOE) activities were not uniform among adult age classes, suggesting that maturus junior, maturus, and maturus senior differentially respond and/or have different susceptibility to ROS. On the other hand, GSH concentration in embryos was undetectable, highest in adolescens and decreased in the latter part of life. Pearson correlation analysis in embryos showed that the activities of the AOEs were strongly and positively correlated with each other but negatively correlated with GSH and SH groups. At later age classes, SOD, CAT, GSH-Px, GR, GSH, and SH groups were no longer significantly correlated with GST. In the discriminant analysis, the variables that separated the age classes were GR, GST, SH groups, and body length. Body length was directly related to the age of individuals, clearly indicating that development/aging affects the regulation of antioxidant defense in this species.

Prooxidant effects of chronic exposure to deltamethrin in green toad Bufotes viridis.

Pesticide-induced oxidative stress, as one of mechanism of toxicity, has been a focus of toxicological research. However, there is a lack of data for certain pesticides-oxidative stress effects especially on terrestrial amphibians. This study evaluates the prooxidative effects of orally administered insecticide deltamethrin (DM) in some tissues of the terrestrial toad Bufotes viridis. Toads were randomly divided and assigned to a control group and a test group that was orally exposed to the pesticide (5 mg/kg of body weight/daily) for 21 days. Animals were euthanized from each group on days 7, 14, and 21, and the liver, leg muscle, ventral skin, and gastrointestinal tissue (GIT) were dissected and used for analysis. From battery of investigated antioxidant components, superoxide dismutase (SOD) was the most differentiate parameter in all examined tissues. For the period of prolonged exposure to pesticide, antioxidative strategy of Bufotes viridis was based on SOD utilization in attempt to maintain the oxidative disbalance at acceptable level. The integrated biomarker response (IBR) as the measure of the overall biochemical response to DM exposure revealed that the group exposed for 21 days had the highest response. Our work has offered valuable data ensuring evidence that toads exposed to deltamethrin developed adaptive reactions that were tissue-specific and that DM-generated systemic toxicity was time-dependent. The present work showed that oxidative stress has significant role in pesticide-induced toxicity and contributes to better understanding of ecotoxicological risk in the terrestrial amphibians exposed to DM.

Oxidative stress parameters in two Pelophylax esculentus complex frogs during pre- and post-hibernation: Arousal vs heavy metals.

In spring, frogs from temperate regions are faced with arousal-induced oxidative stress and exposure to various xenobiotics from the environment. The question is whether pollutants can significantly modify the antioxidative defense system (AOS) response of hibernators during recovery from hibernation. If this assumption is true, we would then expect different patterns of seasonal variations in the AOS between individuals exposed to different levels of pollution. To examine this assumption, we determined the relationship between seasonal variations of accumulated metals and AOS parameters in the skin and muscle of two frog species from the Pelophylax esculentus complex (P. ridibundus and P. esculentus) inhabiting two localities (the Danube-Tisza-Danube canal and the Ponjavica River) with different levels of pollution during pre- and post-hibernation periods, respectively autumn and spring. Our results showed that even though there were differences in the concentrations of accumulated metals and AOS parameters between localities and species, the frogs displayed almost the same patterns of AOS variations during seasons, with a higher AOS response observed in spring. The parameters SH groups, GSH, GR and SOD had been contributed most rather than others. Our findings indicate that oxidative stress during the post-hibernation period was mainly caused by the organisms' recovery from hibernation, as the result of natural selection acting on the AOS, and that the accumulated metals did not significantly modify the AOS response. The present study provides new insight into the biological and physiological cellular responses of frogs to arousal stress.

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. © 2017 SETAC.

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. Fruska 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.

Seasonal variations of cytosolic antioxidant enzyme activities in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) from the Adriatic Sea.

The activity of cytosolic antioxidative defence enzymes in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) were compared in winter and spring in the Adriatic Sea. Activity of antioxidative enzymes is functionally organized due to metabolic demands: analyses of variance and correlation analysis revealed tissue- and seasonal- specific organization of antioxidative enzymes. In winter GST activity increased in both tissues compared with spring. At the same time decreased GSH-Px and GR activities were observed and this effect was more pronounced in liver then in white muscle. From correlation analyses it is concluded that the antioxidative components correlate, but the composition of the antioxidative defence system is different in respect to season and tissue. This means that the antioxidative defence system reorganizes its structure due to oxidative demands and to protect the tissues against reactive oxygen species and to establish homeostasis. Discriminant analyses separated groups according to the complete organization of individual components of the system very well and identified individual components (CAT, GST and GR) which contribute most to the differences. Statistical differences were observed between enzyme activities in tissues (liver and muscle) in both winter and spring, and between seasons (winter and spring) for liver tissue only. Since environmental parameters, such as temperature and oxygen concentration in the sea differ with season, we conclude that in this species the tissues examined expressed their antioxidative defence systems in different ways in respect of external/environmental conditions. We propose that tissue- and seasonal- specific levels of antioxidant enzyme activities should be considered in the interpretation of data from future biomonitoring field studies, especially in relation to low temperature.

[Activity of antioxidative defense enzymes in the blood of patients with liver echinococcosis].

BACKGROUND/AIM: Chronic echinococcocal disease is the parasite human disease caused by the penetration of larval (asexual) stages of the canine tapeworm (Echinococcus granulosus) in the liver of humans. After the penetration of the parasite, the host organism react by activating complement-depending immune response. The aim of this study was to elucidate the influence of larval form of Echinococcus granulosus in the liver on the activity of antioxidative defense enzymes in the blood of patients before and after the surgical intervention. METHODS: We investigated the activity of antioxidative defense enzymes: copper/zinc containing superoxide dismutase (CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione-S-transferase (GST) in the blood of patients before and after the surgical intervention in respect to the controls, clinically healthy persons. RESULTS: Our results showed that the activity of the GSH-Px was significantly decreased in the plasma of the patients with echinocococal disease before the surgery in respect to the controls. The activity of GST was significantly higher in the blood of the patients after the surgery in comparison to the controls. CONCLUSION: Chronic liver echinoccocal disease caused significant changes of some antioxidative defense enzymes, first of all Se-dependent enzyme GSH-Px, which could be a suitabile biomarker in the biochemical evaluation of the disease. This work represents a first comprehensive study of the activity of antioxidative defense enzymes in cronic liver echinococcocosis in the patients before and after the surgical intervention in respect to the clinically healthy persons.