Neuro-immunohistochemical and molecular gene expression variations during hibernation and activity phases between Rana mascareniensis and Rana ridibunda.

Low temperatures and the lack of food during the winter lead the marsh frog Rana ridibunda and the grass frog Rana mascareniensis to hibernate in order to survive. The present study aimed to investigate the cytoarchitecture of brain sub-regions affected by the thermal cycle's fluctuations during the hibernation and activity period, besides the regional distribution quantitative expression of Na(+)/K(+)-ATPase and Pax6 transcriptional factor, the molecular gene expressions of some heat shock proteins, uncoupling protein, and metallothionein. The two frog species were isolated from the field during summer and hibernation time in winter. During hibernation it was notable the destitution of degenerated, pyknotic and vasogenic neurons in different brain areas with high rate nearby the pallium. The immunohistochemical expression of Na+/ K+-ATPase and Pax 6 is decreased during hibernation in different brain sub-regions in the two species suggesting their tendency for energy conservation strategy during hibernation. Additionally, RT-qPCR recorded the up regulation of a number of heat shock protein genes during hibernation with sharing increase between two species for hsp90 besides and the non-significant expression in summer and hibernation periods for hsp47 for both species. Moreover, uncoupling protein (ucp1and ucp2) and metallothionein genes in olfactory bulb were with significant up regulation during the hibernation suggesting that these proteins possibly have a protective effect against reactive oxygen species ROS. So, brain adaptations to low temperature play a crucial role in coordinating stress responses. The present study shed light on the importance of the olfactory bulb in the thermoregulation and sensation of temperature elevations during the hibernation period and defended by the expression of heat shock proteins and uncoupling proteins preventing the cellular damage and proteins misfolding. Neuronal energy production and regeneration activities among amphibians are markedly reduced with decreasing body temperature.

EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond.

Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.

Calcium paradox induces apoptosis in the isolated perfused Rana ridibunda heart: involvement of p38-MAPK and calpain.

"Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

Acute effects of fenthion on certain oxidative stress biomarkers in various tissues of frogs (Rana ridibunda).

This study was aimed mainly to assess the effects of fenthion on certain oxidative stress biomarkers in various tissues of frogs (Rana ridibunda). Biomarkers selected for stress monitoring were malondialdehyde (MDA) and antioxidant defense system (ADS) such as reduced glutathione (GSH) level, glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), and superoxide dismutase (SOD) activities in the liver, kidney, heart, and brain of frogs exposed to 10 and 20 ppm dosages of fenthion for 24, 48, 72, and 96 h. The results demonstrate an increase in MDA levels in selected tissues following exposure to both concentrations of fenthion. The ADS, GSH-Px, GST, SOD activities and GSH levels also fluctuated after 24, 48, 72, and 96 h in all the treatment groups compared with controls. From the evidence obtained here, it is concluded that the exposure of frogs to fenthion induced an increase in MDA combined with fluctuated ADS. This may reflect the potential role of these parameters as useful biomarkers for oxidative stress in amphibian species.

Omethoate modulates some oxidant/antioxidant parameters in frogs (Rana ridibunda Pallas).

The aim of this study was to investigate the effect of an organophosphorus insecticide omethoate (OM), on certain oxidative stress biomarkers (malondialdehyde (MDA), reduced glutathione (GSH), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT)) in tongue, lung, stomach and muscle tissues of adult frogs (Rana ridibunda Pallas). Animals were exposed to 10 and 20 parts per million dosages of OM for 24, 48, 72 or 96 h. According to the results, MDA level increased significantly in lung and stomach tissues. GSH content fluctuated in lung and muscle while it elevated in tongue and stomach tissues. With regard to antioxidant enzymes (GST, GR and CAT), their activities reduced in tongue, while they increased in lung and fluctuated in stomach and muscle tissues. It can be concluded that exposure of frogs to OM are characterized by increased MDA levels and fluctuated enzyme activities and GSH contents. This may reflect the potential role of these parameters as useful biomarkers for assessment of OM toxicity.

Immunocytochemical study of NR1, NR2A and NR2B subunits of NMDA receptor in frog retina (Rana ridibunda).

The NMDA receptors are ionotropic glutamate receptors that are involved in a variety of functions in the nervous system and in particular in the retina. They are composed of NR1 and NR2 subunits. The NMDA receptors have been fairly well studied in the retina of mammals, however, there is only limited information concerning these receptors in the retinas of lower vertebrates. The aim of the present study was to investigate immunocytochemically the NR1, NR2A and NR2B subunits of the NMDA receptors in the frog retina. Six primary antibodies were used. Three of them were directed to different splice variants of the NR1 subunit and the remaining three variants directed to NR2 subunits. All antibodies showed well expressed labeling in the frog retina. The labels had a punctate character and were located mainly in the inner and the outer plexiform layers. The results obtained indicate that the NR1, NR2A and NR2B subunits of NMDA receptor may participate in the glutamatergic neurotransmission from photoreceptors to second order retinal neurons, as well as from bipolar cells to third order retinal neurons. It has been proposed that in the frog retina, several subtypes of NMDA receptors exist each involved with different functions.

Validation of oxidative stress responses in two populations of frogs from Western Ukraine.

Although amphibians are considered to be good bioindicators of environmental pollution, few data are available concerning their biochemical parameters in natural populations. We investigated seasonal (spring, summer, autumn) and spatial (wetlands in rural and urban areas) fluctuations of oxidative stress biomarkers in the liver of frog Rana ridibunda in Western Ukraine. The Centroid grouping analysis demonstrated that despite the fluctuations of separate indices, frogs from an urban site in summer and autumn are differed widely from those at the same site in spring and frogs from the rural site in all three seasons, joined in common set. In summer, suppression of Mn-superoxide dismutase and catalase activity, as well as increase in oxidized glutathione and lipid peroxidation levels demonstrate a collapse of antioxidant defense system in frogs from an urban site. The integrated oxidative stress index confirms this conclusion.

Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation.

The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway in tissues of frog Rana ridibunda and land snail species Helix lucorum during seasonal hibernation. Moreover responses of glycolytic enzymes from cold acclimated R. ridibunda and H. lucorum were studied as well. The drop in Po(2) in the blood of hibernated frogs and land snails indicated lower oxygen consumption and a decrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had a differential effect on the glycolyis in the two species studied and also in the tissues of the same species. The activity of l-LDH decreased significantly in the skeletal muscle and heart of hibernated R. ridibunda indicating a low glycolytic potential. Similar biochemical responses were observed in the same tissues during cold acclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, might indicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda, the activities of the enzymes increased and remained at higher levels than those of the prehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating land snails.

Different responses of biochemical markers in frogs (Rana ridibunda) from urban and rural wetlands to the effect of carbamate fungicide.

Laboratory studies were conducted to determine the effects of carbamate fungicide TATTU (mixture of propamocarb and mancozeb, 0.091 mg L(-1)) on biochemical markers of exposure in Rana ridibunda from clean (reference) and polluted sites. The untreated animals from the polluted site had lower Cu,Zn- and Mn-superoxide dismutase (SOD) and acetylcholinesterase activity, the levels of lipid peroxidation products (TBARS) and protein carbonyls in the liver and vitellogenin-like proteins (Vtg-LP) in the serum, but higher levels of glutathione in the liver in comparison with untreated frogs from the reference site. Catalase activity, superoxide anion and metallothionein levels were the same in both groups. The animals from two sites demonstrate different response on the effect of TATTU during 14 days. In the frogs from polluted site the oxidative damage (the decrease of Mn-SOD activity, lipids and protein oxidative destruction), neurotoxicity (depletion of acetylcholinesterase activity), and endocrine disruption (increase of Vtg-LP level) were revealed. On the other hand, the part of the indices in the animals from the reference site was unchanged after the treatment and the level of metallothionein was elevated demonstrating the satisfactory ability for the adaptation to unfavourable conditions.

Seasonal and spatial comparison of metallothioneins in frog Rana ridibunda from feral populations.

The aim of this study was to elucidate the seasonal and spatial regularity of the properties of metallothioneins (MTs) from the liver and kidney of the frog Rana ridibunda in rural (R) and urban (U) sites in Western Ukraine. This allowed examination of their possibility use in biomonitoring of environmental quality. The positive correlation for Zn and negative correlation for Cu were reflected between their content in the liver and MTs. The content of MTs was higher in summer compared to other seasons and also at the U site compare to the R site. MTs had been comprised of two chromatographic forms (MT-1 and MT-2/MT-2a), with lesser and variable MT-2/2a in frogs from the U site, particularly in the kidney. MTs accumulated about 75% of Cd in the liver. In summary, the ability MTs to elevate content as a stress response, together with the sensitivity of MT-2, may be explored to understand the health status of the frog in each season, reflecting the higher overall anthropogenic impact at the U site.

Comparison of metal bioavailability in frogs from urban and rural sites of Western Ukraine.

The seasonal and spatial fluctuations of heavy metals in the liver of the frog Rana ridibunda from a river in Western Ukraine were investigated. Liver weight was seen to increase from spring to summer/autumn, most probably as a result of accumulation of metabolites, especially fat and glycogen. The concentrations of the metals found in the liver of the frog was in the order: Fe>Cu approximately Zn>Mn>Cd. For most metals, the highest concentration was recorded in the frogs inhabiting the urban site. The highest level of Cu in the liver was observed in the spring, in the agricultural site, while the highest level of other metals was observed in the summer. The most probable explanation for the high concentration of Cu in the rural site was that in this wetland there were discharge effluents from fungicides with Cu in their formula. Compared to other metals, the bioavailability of Cu was approximately 1000 times higher. The high concentration of Fe rather reflects its fluctuation in the water. Despite its very low concentration in the water (below the limit of detection), Cd was detected in the liver of frogs inhabiting both sites. This is an indication that tissues accumulate Cd, despite the very low concentration detected in the water. This may be an indication of intermittent exposure of frogs to Cd and possibly of other heavy metals.

Cu2+ and acute thermal stress induce protective events via the p38-MAPK signalling pathway in the perfused Rana ridibunda heart.

In the present study, we investigated the induction of the p38-MAPK signalling pathway by copper, as exemplified by CuCl(2), in the isolated perfused heart of the amphibian Rana ridibunda. We found that p38-MAPK phosphorylation by CuCl(2) occurs in a dose-dependent manner, with maximum activation (8.73+/-1.43-fold relative to control values) attained by perfusion with 500 micromol l(-1) CuCl(2) for 15 min, while this activation sustained even after 60 min of reperfusion with normal bicarbonate buffer. CuCl(2) also induced the phosphorylation of the small heat shock protein 27 (Hsp27) in a p38-MAPK dependent manner, as revealed by experiments using the p38-MAPK inhibitor SB203580. p38-MAPK and Hsp27 phosphorylations were also strongly induced by hyperthermia (42 degrees C), while the simultaneous use of hyperthermia and CuCl(2) had a synergistic effect on p38-MAPK activation. Furthermore, perfusions with the potent antioxidant L-ascorbic acid (100 micromol l(-1)), the antioxidant enzymes catalase (CAT) (150 U ml(-1)) or superoxide dismutase (SOD) (30 U ml(-1)) in the presence of 500 micromol l(-1) CuCl(2) did not attenuate the CuCl(2)-induced p38-MAPK activation, implying that at least the reactive oxygen species (ROS) scavenged by these agents are not implicated in this kinase activation. The p38-MAPK phosphorylation induced by the combined action of CuCl(2) and hyperthermia was partially inhibited by catalase, indicating that hyperthermia possibly activates the kinase through the production of H(2)O(2). Caspase-3, an effector protease of apoptosis, remained inactive in hearts perfused at normal or hyperthermic conditions, in the absence or presence of 500 micromol l(-1) CuCl(2). All the above results suggest that, in the amphibian Rana ridibunda heart, p38-MAPK activation by copper has a possible protective role through the small Hsp27.

Heavy metal accumulation and metallothionein concentration in the frog Rana ridibunda after exposure to chromium or a mixture of chromium and cadmium.

The accumulation of two heavy metals (chromium (Cr) and cadmium (Cd)) in the liver, kidney and gut of Rana ridibunda exposed to Cr or to a mixture of Cr and Cd was investigated. The concentration of metallothioneins (MTs) in the same tissues was also studied. Both metals accumulated mainly in the kidney. Cr accumulation in the liver and gut was not affected by the presence of Cd. Furthermore, Cr concentration in the kidney was doubled when Cd was present. MT concentration did not increase after Cr treatment but it increased two- to six-fold over control values in mixture-exposed frogs, the highest value being observed in the gut. MTs in the gut could act as a barrier preventing ingested heavy metals from entering the blood stream. MT concentration correlated positively with Cd concentrations in both the liver and the gut of mixture-exposed animals.

Assessing the effects of the three herbicides acetochlor, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid on the compound action potential of the sciatic nerve of the frog (Rana ridibunda).

To assess the relative toxicity of the herbicides acetochlor and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the nervous system, the sciatic nerve of the frog (Rana ridibunda) nerve was incubated in saline inside a specially designed recording chamber. This chamber permits monitoring of the evoked compound action potential (CAP) of the nerve, a parameter that could be used to quantify the vitality of the nerve in normal conditions as well as when the nerve was exposed to the compounds under investigation. Thus, when the nerve was exposed to acetochlor, the EC(50) was estimated to be 0.22mM, while for 2,4,5-T the EC(50) was 0.90mM. Using the identical nerve preparation, the EC(50) of 2,4-D was estimated to be 3.80mM [Kouri, G., Theophilidis, G., 2002. The action of the herbicide 2,4-dichlorophenoxyacetic acid on the isolated sciatic nerve of the frog (Rana ridibunda). Neurotoxicol. Res. 4, 25-32]. The ratio of the relative toxicity for acetochlor, 2,4,5-T and 2,4-D was found to be 1:4:17.2. However, because it is well-known that the action of 2,4-D is dependent on the pH, the relative toxicity of the three compounds was tested at pH 3.3, since it has been found that the sciatic nerve of the frog is tolerant of such a low pH. Under these conditions, the EC(50) was 0.77mM (from 0.22mM at pH 7.2) for acetochlor, 0.20mM (from 0.90mM) for 2,4,5-T and 0.24mM (from 3.80mM at pH 7.2) for 2,4-D. Thus, the relative toxicity of the three compounds changed drastically to 1:0.25:0.31. This change in the relative toxicity is due not only to the increase in the toxicity of 2,4,5-T and 2,4-D at low pH levels, but also to the decrease in the toxicity of acetochlor at pH 3.3.

Extracellular pH changes activate the p38-MAPK signalling pathway in the amphibian heart.

We investigated the activation of the p38-MAPK signalling pathway during extracellular pH changes in the isolated perfused amphibian heart. Extracellular alkalosis (pH 8.5 or 9.5) maximally activated p38-MAPK within 2 min (4.17- and 3.20-fold, respectively) and this effect was reversible since the kinase phosphorylation levels decreased upon reperfusing the heart with normal Tris-Tyrode's buffer. Extracellular acidosis also activated p38-MAPK moderately, but persistently (1.65-fold, at 1 min and 1.91-fold, at 60 min). The alkalosis-induced p38-MAPK activation depended upon the Na(+)/H(+) exchanger (NHE) and Na(+)/K(+)-ATPase, because it was abolished when the NHE inhibitors amiloride and HOE642 and the Na(+)/K(+)-ATPase inhibitor, ouabain, were used. Our studies also showed that extracellular alkalosis (pH 8.5) induced MAPKAPK2 phosphorylation (2.59-fold, 2 min) and HSP27 phosphorylation (5.33-fold, 2 min) in a p38-MAPK-dependent manner, as it was inhibited with 1 micromol l(-1) SB203580. Furthermore, immunohistochemical studies of the phosphorylated forms of p38-MAPK and HSP27 revealed that these proteins were localised in the perinuclear region and dispersedly in the cytoplasm of ventricular cells during alkalosis. Finally, alkalosis induced the increase of HSP70 protein levels (1.52-fold, 5 min), but independently of p38-MAPK activation. These data indicate that the p38-MAPK signalling pathway is activated by extracellular pH changes and in the case of alkalosis this activation may have a protective role.

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues.

The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl(2) L(-1) for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3-mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium.

Survival and metabolic responses to freezing by the water frog (Rana ridibunda).

We studied the ability of the marsh frog Rana ridibunda to survive freezing exposure and the associated subsequent metabolic variations. This species that typically overwinters under water tolerates the conversion of 55% of its body water into ice. This ice content is attained after a few hours (between 8 and 36 hours depending on the mass of the individual and the environmental temperature) but death occurs at greater than 58% ice. Freezing stimulated a significant increase in blood carnitine and trimethylamine levels (respectively 4.5+/-2.5 and 0.5+/-0.2 micromol.l(-1) for controls versus 27.0+/-18.9 and 3.6+/-4.1 micromol.l(-1) after thawing) but these increases had no significant effect on plasma osmolality which was unchanged between control and freeze exposed frogs (252.6+/-20.3 versus 240.2+/-25.0 mOsmol.l(-1), respectively). Freezing also induced a significant dehydration of heart, liver and muscles (respectively 4.2, 3.2 and 2.8%) but the observed levels are low compared to values found in highly freeze tolerant species. This species could be classified as "partially freeze tolerant" enduring the transformation of a significant part of its body water into ice but not the completion of the exotherm. The existence of freeze tolerance in an aquatic hibernator that does not accumulate cryoprotectant, exhibiting low organ dehydration after freezing and low hypoxia tolerance, raises the possibility that a tolerance of nearly 60% ice within the body is common among anurans.

Activation of Na+/H+ exchange by protein phosphatase inhibitors in red blood cells of the frog Rana ridibunda.

The present study was designed to evaluate the role of protein phosphatases in regulation of sodium transport in the marsh frog erythrocytes using 22Na as a tracer. For this purpose the cells were treated with several known inhibitors of protein phosphatases. In standard isotonic medium, exposure of the cells to 10 mmol l(-1) NaF, 20 nmol l(-1) calyculin A or 0.1 mmol l(-1) cantharidin resulted in a significant (1.7-fold) increase in unidirectional ouabain-insensitive Na+ influx. The Na+ influx in frog red cells was progressively activated as the medium osmolality was increased by addition of 100, 200 or 300 mmol l(-1) sucrose to standard isotonic medium. The stimulatory effect of protein phosphatase blockers on Na+ influx was much higher in hypertonic medium containing 100 or 200 mmol l(-1) sucrose than that in isotonic medium. Stimulation of Na+ transport enhanced with increasing concentrations of calyculin A, and half-maximal activation (EC50) was obtained at 16 nmol l(-1). However, Na+ influx induced by strong hypertonic treatment (+300 mmol l(-1) sucrose) was not altered further in the presence of protein phosphatase inhibitors. The changes in Na+ influx evoked by protein phosphatase inhibitors and hypertonic treatment were associated with a rise in the intracellular Na+, but not K+, content. Enhancement in Na+ influx after addition of protein phosphatase blockers to cell suspension in isotonic or hypertonic media was almost completely inhibited by Na+/H+ exchange inhibitors, amiloride and ethyl-isopropyl-amiloride. The basal Na+ influx in frog erythrocytes in isotonic medium was relatively low (1.7 mmol/l cells/h) and not affected by 1 mmol l(-1) amiloride. Thus, the data obtained clearly indicate that Na+/H+ exchanger in the marsh frog red blood cells is under tight regulatory control, in all likelihood via protein phosphatases of types PP-1 and PP-2A.