Frog Liver Research Articles

Changes in Liver Parenchyma of Green Frogs (Pelophylax Esculentus Complex) Under Conditions of Anthropogenic Pollution and Their Use in Monitoring of Water Bodies

Determination of the total biological effects of anthropogenic water pollution remains an important issue. Our long-term studies have shown that numerous alterations occur in the frog liver histological structure under pressure of anthropogenic pollution factor. Th e leukocyte infiltration, fat dystrophy, protein dystrophy and necrosis are well known on the mammal liver. Also we first described the rebuilding in the normal liver structure and the depletation of the fibrous tissue which are characteristic for the amphibians. Quantitative analysis of these alterations can identify significant differences in the pattern of pathological changes in the liver of the green frogs, which pick up in anthropogenically contaminated landscapes and in the clear ponds. Th is method allows quantifying the degree of biological effect of pollution.

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.

Seasonal variation and freezing response of glucose transporter 2 in liver of the wood frog: implications for geographic variation in freeze tolerance

AbstractSubarctic populations of the wood frog Rana sylvatica survive freezing to temperatures at least 10–13°C below those of more southerly conspecifics. This profound freeze tolerance is due in part to an enhanced glucosic cryoprotectant system that requires rapid mobilization of glucose from hepatocytes during the early hours of freezing. To determine if glucose transporter 2 (GLUT2) in the liver contributes to geographic variation in freeze tolerance, we examined changes in the protein's abundance seasonally and in response to experimental freezing in frogs from Interior Alaska and southern Ohio, USA. Using immunoblotting techniques, we found that GLUT2 abundance increased in preparation for winter in both populations, but tests with Ohioan frogs showed that that altered temperature alone does not cause these seasonal changes. In Ohioan frogs, transporter expression apparently was regulated transcriptionally, as mRNA levels, assessed using quantitative real‐time polymerase chain reaction, changed in accordance with protein abundance. However, this pattern was not observed in Alaskan frogs, suggesting that other mechanisms of regulation are important in this phenotype. Overall, GLUT2 abundance was constitutively greater and more responsive to freezing in Alaskan R. sylvatica, suggesting that GLUT2 contributes substantively to the extreme freeze tolerance of subarctic wood frogs.

Hepatic metallothioneins in molecular responses to cobalt, zinc, and their nanoscale polymeric composites in frog Rana ridibunda

Despite numerous studies suggesting a dramatic decline of amphibians, the biochemical mechanisms of adaptation in these animals to polluted environment are poorly studied. The aim of this study was to elucidate the ability to release cobalt (Co) and zinc (Zn) from their nanoscale complexes (NCs) derived from the polymeric substance of N-vinylpyrrolidone (PS) in the liver of amphibian (Rana ridibunda). Frog males were subjected to 14days exposure to waterborne Co2+ (50μg/L), Zn2+ (100μg/L), as well as corresponding concentrations of Co-NC, Zn-NC or PS. Main attention was paid to MT's interrelations with indices of stress and toxicity. Only Co2+ and Zn2+ caused elevation of the correspondent metal in MTs. Co2+ caused down-regulation of cathepsin D activity, while Zn2+, Zn-NC and the PS up-regulated this activity. Zn2+ provoked 1.6 times increase of metal-bounded form of the MT (MT-Me), while all other exposures caused the elevation of the ratio of MT total protein concentration (MT-SH) and concentrations of the MT-Me and/or immunoreactive (MTi) form (up to ~10 times) accompanied by a decrease in the levels of oxyradicals. The increased DNA fragmentation and down-regulation of caspase-3 activity in relation to the redox state of glutathione and/or lactate/pyruvate were shown at all exposures. These data indicate the vulnerability of the redox state of cellular thiols and inability to release Co and Zn from NCs in frog's liver.

Heavy metal mediated immunomodulation of the Indian green frog, Euphlyctis hexadactylus (Anura:Ranidae) in urban wetlands

Impacts of heavy metal toxicity on the immune system of the Indian green frog, Euphlyctis hexadactylus, in Bellanwila Attidiya, an urban wetland polluted with high levels of heavy metals, compared to the reference site in Bolgoda, in Sri Lanka was investigated. Significantly higher accumulation of selected heavy metals, copper (Cu), zinc (Zn), lead (Pb), and cadmium (Cd) were detected by AAS in frog liver and gastrocnemius muscle, in the polluted site than in the reference site. Non-functional immunotoxicity tests; total WBC, splenocyte and bone marrow cell counts, spleen weight/body weight ratio, neutrophil/lymphocyte ratio and basal immunoglobulin levels, and phagocytic capacity of peritoneal macrophages (immune functional test) were carried out using standard methodology. Test parameters recorded significantly lower values for frogs of the polluted site compared with their reference site counterparts, indicative of lowered immune response of frogs in the former site. In vitro phagocytic assay based on NBT dye reduction, measured the stimulation index (SI) of E. hexadactylus blood leukocytes, splenocytes and peritoneal macrophages, where SIs of frogs in the polluted site were significantly lower. Also, in vitro exposure of frog phagocytes to Cu, Zn, Pb and Cd at 10−2–10−10M, showed immunomodulation i.e. low concentrations stimulated phagocytosis while increased concentrations showed a trend towards immunosuppression. IC50 values indicated Cd>Zn>Cu>Pb as the decreasing order of the potential of phagocytosis inhibition. In conclusion, this study clearly demonstrated immunomodulation of E. hexadactylus, stimulated by heavy metals. In-vitro studies evidently suggested the use of phagocytosis as a biomarker in Ecoimmunotoxicology to detect aquatic heavy metal pollution.

Chronic exposures to monomethyl phthalate in Western clawed frogs

Polymer flexibility and elasticity is enhanced by plasticizers. However, plasticizers are often not covalently bound to plastics and thus can leach from products into the environment. Much research effort has focused on their effects in mammalian species, but data on aquatic species are scarce. In this study, Western clawed frog (Silurana tropicalis) embryos were exposed to 1.3, 12.3, and 128.7mg/L monomethyl phthalate (MMP) until the juvenile stage (11weeks) and to 1.3mg/L MMP until the adult stage (51weeks). MMP decreased survival, hastened metamorphosis, and biased the sex ratio toward males (2M:1F) at the juvenile stage without altering the expression of a subset of thyroid hormone-, sex steroid-, cellular stress- or transcription regulation-related genes in the juvenile frog livers. At the adult stage, exposure to MMP did not have significant adverse health effects, except that females had larger interocular distance and the expression of the heat shock protein 70 was decreased by 60% in the adult liver. In conclusion, this study shows that MMP is unlikely to threaten amphibian populations as only concentrations four orders of magnitude higher than the reported environmental concentrations altered the animal physiology. This is the first complete investigation of the effects of phthalates in a frog species, encompassing the entire life cycle of the organisms.

Effect of physiological stress on expression of glucose transporter 2 in liver of the wood frog, Rana sylvatica.

Glucose transporters (GLUTs) have been implicated in the survival of various physiological stresses in mammals; however, little is known about the role of these proteins in stress tolerance in lower vertebrates. The wood frog (Rana sylvatica), which survives multiple winter-related stresses by copiously mobilizing hepatic glycogen stores, is an interesting subject for the study of glucose transport in amphibians. We examined the effects of several physiological stresses on GLUT2 protein and mRNA levels in the liver of R. sylvatica. Using immunoblotting techniques to measure relative GLUT2 abundance, we found that GLUT2 numbers increased in response to organismal freezing, hypoxia exposure, and glucose loading; whereas, experimental dehydration and urea loading did not affect GLUT2 abundance. GLUT2 mRNA levels, assessed using quantitative real-time polymerase chain reaction, changed in accordance with protein abundance for most stresses, indicating that transcriptional regulation of GLUT2 occurs in response to stress. Overall, hepatic GLUT2 seems to be important in stress survival in R. sylvatica and is regulated to meet the physiological need to accumulate glucose.

P09 : Regulation of mTOR complex 1 in Liver of Freeze Tolerant wood frogs, Rana sylvatica

Many organisms experience seasonal cold environments during their life and need specific adaptations to allow them to survive when the ambient temperature fall below 0 °C. Previous studies showed a high survival rate (∼100%) of wood frog during different conditions of freezing exposure (various time and/or temperature freezing, and developmental stages of the animals). In order to balance ATP synthesis and consumption under the cold condition, optional yet energy expensive processes such as cell growth need to be suppressed or inhibited. The rate-limiting step of mRNA translation is the initiation stage, which largely regulated by the kinase complex mTORC1 (mTOR complex 1). Active mTORC1 phosphorylates the translation inhibitor 4EBP (eIF4E binding protein) and promoter S6K (40S ribosomal protein S6 kinase). Hyperphosphorylation of 4EBP inhibits its interaction with eIF4E, thereby relieving the inhibitory effect on translation pre-initiation complex assembly. Phosphorylated S6K by mTORC1 is able to enhance the RNA helicase activity of eIF4A by phosphorylating eIF4B, to clear the inhibitory secondary structures of mRNA for translation initiation machinery to reach start codon.

Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis

The African clawed frog, Xenopus laevis, is able to withstand extremely arid conditions by estivating, in conjunction with dehydration tolerance and urea accumulation. Estivating X. laevis reduce their metabolic rate and recruit anaerobic glycolysis, driven by lactate dehydrogenase (LDH; E.C. 1.1.1.27) enzymes that reversibly convert pyruvate and NADH to lactate and NAD(+), to meet newly established ATP demands. The present study investigated purified LDH from the liver of dehydrated and control X. laevis. LDH from dehydrated liver showed a significantly higher K m for L-lactate (1.74 fold), NAD(+) (2.41 fold), and pyruvate (1.78 fold) in comparison to LDH from the liver of control frogs. In the presence of physiological levels of urea found in dehydrated animals, the K m values obtained for dehydrated LDH all returned to control LDH K m values. Dot blot analysis showed post-translational modifications may be responsible for the kinetic modification as the dehydrated form of LDH showed more phosphorylated serine residues (1.54 fold), less methylated lysine residues (0.43 fold), and a higher level of ubiquitination (1.90 fold) in comparison to control LDH. The physiological consequence of dehydration-induced LDH modification appears to adjust LDH function in conjunction with urea levels in dehydrated frogs. When urea levels are high during dehydration, LDH retains its normal function. Yet, as urea levels drop during rehydration, LDH function is reduced, possibly shunting pyruvate to the TCA cycle.

Bioavailability and tissue distribution of Dechloranes in wild frogs (Rana limnocharis) from an e-waste recycling area in Southeast China

Dechlorane Plus (DP), a flame retardant used as an alternative to decabromodiphenylether, has been frequently detected in organisms, indicating its bioaccumulation and biomagnification potential in aquatic and terrestrial species. However, little data is available on the bioaccumulation of DP in amphibians. Dechlorane Plus and its analogs (DPs) were detected in the liver, muscle and brain tissues of wild frogs (Rana limnocharis), which were collected from an e-waste recycling site, Southeast China. DP, Mirex, Dec 602 and a dechlorinated compound of DP (anti-Cl11-DP) varied in the range of 2.01–291, 0.650–179, 0.260–12.4, and not detected (nd)–8.67 ng/g lipid weight, respectively. No difference of tissue distribution was found for syn-DP, Mirex and Dec 602 between the liver and muscle tissue (liver/muscle concentration ratio close to 1, p > 0.05). However, higher retention was observed for anti-DP and anti-Cl11-DP in the frog muscle relative to the liver tissue (liver/muscle concentration ratio < 1, p < 0.05). Additionally, the blood-brain barrier was found to work efficiently to suppress these compounds entering brain tissues in this species (liver/brain concentration ratio > 1, p < 0.05), and the molecular weight was a key factor impacting the extent of the blood-brain barrier. Compared to levels in the muscle and brain tissue, a preferential enrichment of syn-DP was observed in the liver tissue, suggesting the occurrence of stereo-selective bioaccumulation in the wild frog.

149 DNA methylation and regulation of DNMTs in freeze tolerant wood frog, Rana sylvatica

Wood frog ( Rana sylvatica ) is one of the few vertebrate species that have perfected the biological adaptations of surviving whole-body freezing when the temperature falls below 0 °C in its natural habitat. It has been established that the metabolic rate is coordinated at different stages of the whole-body freezing of wood frog. Energy expensive and optional biological processes need to be repressed or completely shut down; while stress responsive pathways are activated. For example, overall gene transcription is largely suppressed during hypometabolism. This can be achieved at a genomic level via methylation at the C-5 position on cytosine residue of DNA (5mC). The present study explores DNA methylation in wood frog freezing survival by examining the protein expression levels and overall DNA methyltransferase activities of the major DNMTs in liver and muscle of both frozen and glucose-loaded wood frogs. The overall levels of 5′-methylcytosine and 5′-hydroxycytosine in muscle and liver genomic DNA are also compared between stress/glucose-loaded and control frogs. The results showed that freezing exposure led to increased protein levels of selected DNMTs in muscle ad liver of wood frog. In addition, the 5mC level dropped in liver but increased in muscle upon freezing exposure. The DNMT activity showed consistent changes in both tissues. Our data also suggest that high glucose (∼200 mM) is able to inhibit overall DNMT activity in both tissues of wood frog. The present study demonstrated that response of DNMT-dependent DNA methylation is an integral part of wood frog freeze-tolerance. Source of funding: None declared. Conflict of interest: None declared. kenneth_storey@carleton.ca

Oxidative damage and apoptosis induced by microcystin-LR in the liver of Rana nigromaculata in vivo

Microcystins (MCs) are hepatotoxins with potent inhibitor activity of protein phosphatases PP1 and PP2A. The present study shows that MC-LR can induce severe oxidative damage and apoptosis in the livers of frogs (Rana nigromaculata) exposed to 1μg/L MC-LR for 7 and 14d in vivo. Ultrastructural observation showed the apoptotic morphology of perinuclear chromatin margination and swollen mitochondria, indicating that MC-LR can significantly damage frog liver. Reactive oxygen species (ROS) production and malondialdehyde (MDA) content were positively correlated with exposure time. Meanwhile, reduced glutathione (GSH) content and GSH peroxidase (GPx) activity rapidly decreased after prolonged exposure to 1μg/L MC-LR in a time-dependent manner. These results imply that the antioxidant defense systems of the liver were damaged. Enhanced apoptosis of cells in the livers of MC-treated frogs was detected by terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the mitochondrial system. MC-LR significantly stimulated the livers to release cytochrome c, which improved the protein expressions of Bax, caspase-3, and caspase-9 (p<0.01) and inhibited the protein expression of Bcl-2 with prolonged exposure (p<0.01) via the mitochondrial pathway. These results imply that the mitochondrial pathway has a key function in toxin-induced liver cell apoptosis. The expression of caspase-8 was induced significantly (p<0.01), which illustrates the mechanism that the death receptor pathway is also involved in apoptosis. The present findings show that MC-LR can induce apoptosis in frog liver, which may be related with the decline of amphibian populations. The World Health Organization-recommended drinking water limit for MC-LR in water may be not safe for amphibians.

Changes in Activity of Three Sulfurtransferases in Response to Exposure to Cadmium, Lead and Mercury Ions

Cadmium, lead and mercury are environmentally persistent toxicants that affect tissues and cellular components or exert an effect on generation of reactive oxygen species causing a decreased level of available antioxidant reserves. Sulfurtransferases are enzymes that are widespread in nature. Rhodanese, 3-mercaptopyruvate sulfurtransferase and γ-cystathionase play an important role in the metabolism of L-cysteine. Heavy metal ions can bind to -SH groups of cysteine residues in their active sites and, therefore, decrease the activity of these enzymes and result in changes in the level of sulfane sulfur-containing compounds, products of L-cysteine desulfuration. Changes in the activity of sulfurtransferases were investigated in the kidneys, heart, brain, liver and skeletal muscle of Marsh frogs (Pelophylax ridibundus) after 10 days of exposure to Pb(NO3)2 at the concentration of 28 mg/L and CdCl2 at the concentration of 40 mg or 80 mg/L, and in Xenopus laevies tissues after 7 and 14 days of exposure to HgCl2 at the concentration of 1.353 mg/L. The investigated heavy metal ions have a tendency to inhibit the activity of sulfurtransferases and decrease the level of glutathione, what can result in oxidative stress and oxidation of cysteine -SH groups to -SOH. This reversible oxidation and reduction of these redox sensitive groups can play a role in defenses against oxidative stress. Based on the presented results, one can surmise that also the expression of the three sulfurtransferases depends on heavy metal ions and/or some parameters of oxidative stress, what can explain the increase of the activity of MPST and CST in the kidney.

Hepatic carbamoyl phosphate synthetase (CPS) I and urea contents in the hylid tree frog, Litoria caerulea: transition from CPS III to CPS I

The complete cDNA sequence of CPS I obtained from the liver of the hylid tree frog, Litoria caerulea, consisted of 4,485 bp which coded for 1,495 amino acids with an estimated molecular mass of 163.7 kDa. The deduced CPS I consisted of a mitochondrial targeting sequence of 33 amino acid residues, a glutaminase amidotransferase component spanning from tyrosine 95 to leucine 425, and a methylglyoxal synthetase-like component spanning from valine 441 to lysine 1566. It also comprised two cysteine residues (cysteine 1360 and cysteine 1370) that are characteristic of N-acetyl-L-glutamate dependency. Similar to the CPS I of Rana catesbeiana and Cps III of lungfishes and teleosts, it contained the Cys-His-Glu catalytic triad (cysteine 304, histidine 388 and glutamate 390). All Cps III contain methionine 305 and glutamine 308, which are essential for the Cys-His-Glu triad to react with glutamine, but the CPS I of R. catesbeiana contains lysine 305 and glutamate 308, and therefore cannot effectively utilize glutamine as a substrate. However, the CPS I of L. caerulea, unlike that of R. catesbeiana, contained besides glutamate 308, methionine 305 instead of lysine 305, and thus represented a transitional form between Cps III and CPS I. Indeed, CPS I of L. caerulea could utilize glutamine or NH₄⁺ as a substrate in vitro, but the activity obtained with glutamine + NH₄⁺ reflected that obtained with NH₄⁺ alone. Furthermore, only <5 % of the glutamine synthetase activity was present in the hepatic mitochondria, indicating that CPS I of L. caerulea did not have an effective supply of glutamine in vivo. Hence, our results confirmed that the evolution of CPS I from Cps III occurred in amphibians. Since L. caerulea contained high levels of urea in its muscle and liver, which increased significantly in response to desiccation, its CPS I had the dual functions of detoxifying ammonia to urea and producing urea to reduce evaporative water loss.

Getting under—and through—the skin: ecological genomics of chytridiomycosis infection in frogs

Amphibian species around the world are currently becoming endangered or lost at a rate that outstrips other vertebrates—victims of a combination of habitat loss, climate change and susceptibility to emerging infectious disease (Stuart et al. 2004). One of the most devastating such diseases is caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), which infects hundreds of amphibian species on multiple continents. While Bd itself has been characterized for some time, we still know little about the mechanisms that make it so deadly. In this issue of Molecular Ecology, Rosenblum et al. describe a genomic approach to this question, reporting the results of a genome-wide analysis of the transcriptional response to Bd in the liver, skin and spleen of mountain yellow-legged frogs (Rana mucosa and R. sierrae: Fig. 1) (Rosenblum et al. 2012). Their results indicate that the skin is not only the first, but likely the most important, line of defence in these animals. Strikingly, they describe a surprisingly modest immune response to infection in Rana, a result that may help explain variable Bd susceptibility across populations and species.

Cell cycle regulation in the freeze tolerant wood frog, Rana sylvatica

The wood frog (Rana sylvatica) is one of only a few vertebrate species that can survive extensive freezing of its body fluids during the winter. The mechanisms of natural freeze tolerance include metabolic rate depression to conserve energy and the implementation of cryoprotective strategies, especially the synthesis of huge amounts of glucose as a cryoprotectant. Liver is the main source of glucose production/export (and other cryoprotective actions) and plays a central role in freezing survival of the whole animal. Freezing is a multi-component stress that includes anoxia/ischemia due to the cessation of blood flow and dehydration of cells caused by ice accumulation in extracellular spaces. To help endure these stresses, cells need to suppress and reprioritize ATP-expensive cell functions. One of these is cell growth and proliferation, and we hypothesized that cell cycle arrest would be key to freezing survival. The present study examines the responses by key cell cycle components to freezing, anoxia and dehydration stresses in wood frog liver. Immunoblotting was used to investigate protein expression of Cdc 2, Cdks (2, 4, 6), and cyclins (A, B1, D1, E) as well as the phosphorylation states of Cdks (Thr14/Tyr15), the phosphatases Cdc25a (Ser76) and Cdc25c (Ser216) and the CIP/KIP Cdk inhibitors p21 (Thr145) and p27 (Thr187). Responses to 24 h freezing, 24 h anoxia and 40% dehydration as well as recovery from these stresses were analyzed. The results showed very similar responses by cell cycle components to anoxia or dehydration and were consistent with cell cycle suppression under stress and reversal during recovery. Freezing showed elements of cell cycle suppression, including reduced protein levels of Cdks and cyclins A and B1, but also showed unique responses by cyclin D1, Cdc25 phosphatases and p21/p27. These may be linked with alternative actions by these proteins that contribute to cryoprotection; e.g., an alternative action of cyclin D1 as a transcription factor may contribute to the upregulation of glucose-6-phosphatase, a key enzyme needed for the export of glucose cryoprotectant.

Exposure to lead in water and cysteine non-oxidative metabolism in Pelophylax ridibundus tissues

Chronic, low-level exposure to metals is an increasing global problem. Lead is an environmentally persistent toxin that causes many lead-related pathologies, directly affects tissues and cellular components or exerts an effect of the generation of reactive oxygen species causing a diminished level of available sulfhydryl antioxidant reserves. Cysteine is one of substrates in the synthesis of glutathione – the most important cellular antioxidant, and it may also undergo non-oxidative desulfuration that produces compounds containing sulfane sulfur atoms. The aim of the experiment was to examine changes of the non-oxidative metabolism of cysteine and the levels of cysteine and glutathione in the kidneys, heart, brain, liver and muscle of Marsh frogs (Pelophylax ridibundus) exposed to 28mg/L Pb(NO3)2 for 10 days. The activities of sulfurtransferases, enzymes related to the sulfane sulfur metabolism – 3-mercaptopyruvate sulfurtransfearse, γ-cystathionase and rhodanese – were detected in tissue homogenates. The activity of sulfurtransferases was much higher in the kidneys of frogs exposed to lead in comparison to control frogs, not exposed to lead. The level of sulfane sulfur remained unchanged. Similarly, the total level of cysteine did not change significantly. The total levels of glutathione and the cysteine/cystine and GSH/GSSG ratios were elevated. Thus, it seems that the exposure to lead intensified the metabolism of sulfane sulfur and glutathione synthesis in the kidneys. The results presented in this work not only confirm the participation of GSH in the detoxification of lead ions and/or products appearing in response to their presence, such as reactive oxygen species, but also indicate the involvement of sulfane sulfur and rhodanese in this process (e.g. brain). As long as the expression of enzymatic proteins (rhodanese, MPST and CST) is not examined, no answer will be provided to the question whether changes in their activity are due to differences in the concentrations of substrates and/or compounds affecting their activity or to changes in their level in response to some parameters, e.g. associated with oxidative stress.

The activity of some enzymes in the frog's liver (Rana ridibunda Pall.) in the different natural conditions of thermal water regime

During the period of March–July 2011, the activity of aspartate aminotranspherase (AST), alanine aminotranspherase (ALT), lactate dehidrogenase (LDH) and alkaline phosphatase (ALP) enzymes was measured in the liver of the Rana ridibunda Pall. natural frog population.Altogether 52 frogs were collected for the research purposes in the two rivers of a different water temperature regime respectively, the thermal water spring of Banja of Malishevë (24.7°C – 29.0°C) and river Tërdec (7.9°C – 19.7°C).There were not recorded great oscillations of AST and ALT activity in the liver of frogs from the thermal water, and ALT activity from the river Tërdec as well during the period of investigation. At the same time in the liver of frogs from the river Tërdec, the lower activity of AST has been recorded in March (12.7±2.6 ), whereas a higher activity in May (41.4±8.4 ), and in July (46.6± 7.6). The distinctive characteristic of this research is that only in March, the activity of AST in the frogs liver from the river Tërdec is significantly at lower level compared with values of frogs from the thermal water.Despite AST, in the liver of the frogs of two localities there were not recorded large oscillations of the ALT, LDH and ALP activity values during the entire period of research.According to the research results we could conclude that the water temperature (the cold stress), the seasonal changes of the environmental temperature, the nutrition, and the metabolic activity of the frogs, affects the activity of AST.

Myxozoan Parasite in Brain of Critically Endangered Frog

Myxozoan Parasite in Brain of Critically Endangered Frog

DDTs in rice frogs (Rana limnocharis) from an agricultural site, South China: Tissue distribution, biomagnification, and potential toxic effects assessment

Contamination with agricultural pesticides such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), is among several proposed stressors contributing to the global declines in amphibian populations and species biodiversity. These chemicals were examined in insects and in the muscle, liver, and eggs of rice frogs (Rana limnocharis) from the paddy fields of an agricultural site in South China. The ΣDDT (sum of DDT, DDE, and DDD) concentrations ranged from 154 to 915, 195 to 1,400, and 165 to 1,930 ng/g lipid weight in the muscle, liver, and eggs, respectively. All the DDTs (DDT, DDE, and DDD) showed higher affinity for the liver relative to muscle tissue and can be maternally transferred to eggs in female frogs. The average biomagnification factors for DDTs ranged from 1.6 to 1.9 and 1.5 to 2.9 in female and male frogs, respectively, providing clear evidence of their biomagnification from insects to frogs. Compared with the reported DDT levels demonstrated to have toxic effects on frogs, DDTs in the present frogs are unlikely to constitute an immediate health risk. However, the adverse impacts of high DDT residues in eggs on the hatching success and their potential toxicity to the newly metamorphosed larval frogs should be assessed further.