Frog Liver Research Articles

The mRNA N6-Methyladenosine Response to Dehydration in Xenopus laevis.

The African clawed frog, Xenopus laevis, exhibits remarkable adaptations to survive in its arid habitat, including behavioral and metabolic changes during periods of drought. During extreme dehydration, X. laevis undergoes estivation, a state characterized by increased urea and ammonia levels, depression of the metabolic rate, and tissue hypoxia. To understand the molecular mechanisms underlying these adaptations, we investigated the potential role of N6-methyladenosine (m6A), a widespread mRNA modification, in X. laevis during extreme dehydration. We analyzed the protein levels of key components in the m6A pathway, including writers (METTL3, METTL14, and WTAP), erasers (ALKBH5 and FTO), and readers (SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a), in the liver and kidneys of control frogs and frogs that had lost 35 ± 0.93% of their total body water. The relative protein levels generally decreased or remained unchanged, with the exception of YTHDF3, which depicted a protein level increase in the liver. Notable changes included eIF3a, which was downregulated by 26 ± 8% and 80 ± 8% in the dehydrated liver and kidney tissues, respectively. Additionally, the total m6A increased by 353 ± 30% and 177 ± 17% in dehydrated liver and kidney RNA samples, respectively. This study highlights the importance of epigenetic mechanisms in stress tolerance and provides a foundation for further exploration of the role of epigenetics in dehydration tolerance.

Biogenesis and Regulation of the Freeze–Thaw Responsive microRNA Fingerprint in Hepatic Tissue of Rana sylvatica

Background: Freeze-tolerant animals undergo significant physiological and biochemical changes to overcome challenges associated with prolonged whole-body freezing. In wood frog Rana sylvatica (now Lithobates sylvaticus), up to 65% of total body water freezes in extracellular ice masses and, during this state of suspended animation, it is completely immobile and displays no detectable brain, heart, or respirometry activity. To survive such extensive freezing, frogs integrate various regulatory mechanisms to ensure quick and smooth transitions into or out of this hypometabolic state. One such rapid and reversible regulatory molecule capable of coordinating many aspects of biological functions is microRNA. Herein, we present a large-scale analysis of the biogenesis and regulation of microRNAs in wood frog liver over the course of a freeze–thaw cycle (control, 24 h frozen, and 8 h thawed). Methods/Results: Immunoblotting of key microRNA biogenesis factors showed an upregulation and enhancement of microRNA processing capacity during freezing and thawing. This was followed with RT-qPCR analysis of 109 microRNA species, of which 20 were significantly differentially expressed during freezing and thawing, with the majority being upregulated. Downstream bioinformatics analysis of miRNA/mRNA targeting coupled with in silico protein–protein interactions and functional clustering of biological processes suggested that these microRNAs were suppressing pro-growth functions, including DNA replication, mRNA processing and splicing, protein translation and turnover, and carbohydrate metabolism. Conclusions: Our findings suggest that this enhanced miRNA maturation capacity might be one key factor in the vital hepatic miRNA-mediated suppression of energy-expensive processes needed for long-term survival in a frozen state.

Proteomic analysis of Rana sylvatica reveals differentially expressed proteins in liver in response to anoxia, dehydration or freezing stress

Ectothermic animals that live in seasonally cold regions must adapt to seasonal variation and specific environmental conditions. During the winter, some amphibians hibernate on land and encounter limited environmental water, deficient oxygen, and extremely low temperatures that can cause the whole body freezing. These stresses trigger physiological and biochemical adaptations in amphibians that allow them to survive. Rana sylvatica, commonly known as the wood frog, shows excellent freeze tolerance. They can slow their metabolic activity to a near halt and endure freezing of 65–70% of their total body water as extracellular ice during hibernation, returning to normal when the temperatures rise again. To investigate the molecular adaptations of freeze-tolerant wood frogs, a comprehensive proteomic analysis was performed on frog liver tissue after anoxia, dehydration, or freezing exposures using a label-free LC–MS/MS proteomic approach. Quantitative proteomic analysis revealed that 87, 118, and 86 proteins were significantly upregulated in dehydrated, anoxic, and frozen groups, suggesting potential protective functions. The presence of three upregulated enzymes, glutathione S-transferase (GST), aldolase (ALDOA), and sorbitol dehydrogenase (SORD), was also validated. For all enzymes, the specific enzymatic activity was significantly higher in the livers of frozen and anoxic groups than in the controls. This study reveals that GST, ALDOA, and SORD might participate in the freeze tolerance mechanism by contributing to regulating cellular detoxification and energy metabolism.

DNA hypomethylation in wood frog liver under anoxia and dehydration stresses

Wood frogs are freeze-tolerant vertebrates that can endure weeks to months frozen during the winter without breathing and with as much as 65% of total body water frozen as extracellular ice. Underlying tolerances of anoxia and of cellular dehydration support whole body freezing. One pro-survival mechanism employed by these frogs is epigenetic modifications via DNA hypomethylation processes facilitating transcriptional repression or activation. These processes involve proteins such as DNA Methyltransferases (DNMTs), Methyl Binding Domain proteins (MBDs), Ten-Eleven Translocases (TETs), and Thymine Deglycosylase (TDG). The present study evaluates the responses of these proteins to dehydration and anoxia stresses in wood frog liver. DNMT relative protein expression was reduced in liver, but nuclear DNMT activity did not change significantly under anoxia stress. By contrast, liver DNMTs and nuclear DNMT activity were upregulated under dehydration stress. These stress-specific differences were speculated to arise from Post-Translational Modifications (PTMs). DNMT3A and DNMT3B showed increased relative protein expression during recovery from dehydration and anoxia. Further, MBD1 was elevated during both conditions suggesting transcriptional repression. TET proteins showed varying responses to anoxia likely due to the absence of oxygen, a main substrate required by TETs. Similarly, TDG, an enzyme that corrects DNA damage, was downregulated under anoxia potentially due to lower levels of reactive oxygen species that damage DNA, but levels returned to normal during reperfusion of oxygen. Our results indicate differential stress-specific responses that indicate the need for more research in the DNA hypomethylation mechanisms employed by the wood frog during stress.

Cytochrome P450 enzymes in the black-spotted frog (Pelophylax nigromaculatus): molecular characterization and upregulation of expression by sulfamethoxazole.

Cytochrome P450 (CYP) enzymes are crucial for the detoxification of xenobiotics, cellular metabolism, and homeostasis. This study investigated the molecular characterization of CYP enzymes in the black-spotted frog, Pelophylax nigromaculatus, and examined the regulation of CYP expression in response to chronic exposure to the antibiotic sulfamethoxazole (SMX) at various environmental concentrations (0, 1, 10, and 100μg/L). The full-length cDNA of Pn-CYP26B1 was identified. The sequence included open reading frames of 1,536 bp, encoding proteins comprising 511 amino acids. The signature motif, FxxGxxxCxG, was highly conserved when compared with a number of selected animal species. SMX significantly upregulated the expression of the protein CYP26B1 in frog livers at concentrations of 1 and 10μg/L. SMX showed an affinity for CYP26B1 of -7.6kcal/mol, indicating a potential mechanism for SMX detoxification or adaptation of the frog. These findings contributed to our understanding of the environmental impact of antibiotics on amphibian species and underscored the importance of CYP enzymes in maintaining biochemical homeostasis under exposure to xenobiotic stress.

The Mechanism of Elizabethkingia miricola Infection of the Black Spotted Frog as Revealed by Multi-Omics Analysis

Elizabethkingia miricola (E. miricola) is a significant pathogen that causes the crooked head disease in black spotted frogs. This disease has plagued numerous frog farms in China and has resulted in substantial losses to the frog farming industry. Nonetheless, the exact mechanism that causes the disease in frogs remains unknown. In this study, transcriptomic and microbiomic analyses were conducted to analyze frog samples infected with E. miricola to reveal the infection mechanism of the pathogen. Liver transcriptomic analysis indicated that the livers of infected frogs had 1469 differentially expressed genes when compared with an uninfected group. These DEGs are mainly involved in immunity and metabolism, including neutrophil extracellular trap formation, the NOD-like receptor signaling pathway, leukocyte transendothelial migration, chemokine signaling pathway, Fc gamma R-mediated phagocytosis, and “metabolism”-related pathways such as the pentose phosphate pathway, carbon metabolism, glycerophospholipid metabolism, and glycerolipid metabolism. Similarly, 4737 DEGs were found in the kidney of infected frogs. These DEGs are mainly involved in immunity, including neutrophil extracellular trap formation, the NOD-like receptor signaling pathway, B cell receptor signaling pathway, C-type lectin receptor signaling pathway, complement and coagulation cascade, and Toll-like receptor signaling pathway. Ten immune-associated DEGs were screened in liver and kidney DEGs, respectively. And it was hypothesized that E. miricola infection could influence the host immune response. Microbiome analysis results showed that some opportunistic pathogens such as Citrobacter, Shigella, and Providencia were significantly elevated (p < 0.05) in infected frogs. Additionally, functional prediction confirmed that most of the microbiota in infected frogs were linked to metabolism-related KEGG pathways. In this study, the screened genes linked to immunity showed an association with the gut microbiome. The majority of these genes were found to be linked with the abundance of opportunistic pathogens. The results showed that E. miricola infection led to the downregulation of immune and metabolic-related genes, which led to the inhibition of immune function and metabolic disorder, and then increased the abundance of opportunistic pathogens in the gut microbiota. The findings of this study offer a preliminary foundation for comprehending the pathogenic processes of E. miricola infection in black spotted frogs.

Surviving winter on the Qinghai-Xizang Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri.

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

THE CATALYTIC PROPERTIES OF LACTATE DEHYDROGENASE IN SKELETAL MUSCLES AND LIVER OF THE MARSH FROG (PELOPHYLAX RIDIBUNDUS) DEPEND ON ITS ECOLOGICAL AND GEOGRAPHICAL LOCATION

The marsh frog (Pelophylax ridibundus) has a widespread distribution range, which is due to a variety of adaptations that contribute to the development of tolerance to a wide range of physicochemical environmental factors. Of particular interest are the adaptations of these animals to different levels of oxygen in mid- and high-altitude conditions. In this work, a comparative analysis of the kinetic parameters of lactate dehydrogenase (LDH) in the liver of marsh frogs living in the mountainous and lowland regions of Dagestan was carried out. Animals caught in their habitats were decapitated, the liver and calf muscles were isolated, and they were placed in liquid nitrogen. In the laboratory, the selected tissues were homogenized and mitochondria-free cytosol was obtained by differential centrifugation, in which LDH activity was determined. It was found that LDH activity is significantly higher in the tissues of frogs from mountainous regions: by 42.4% in the muscles and 2.38 times in the liver (p 0.05). The high efficiency of catalysis is ensured due to significant changes in the catalytic parameters of the enzyme: an increase in Vmax (50.9% in muscles and 70% in the liver (p 0.05)) and a decrease in Km. (45.9% in muscles and 69% in liver, (p 0.05)). A more pronounced difference, compared to muscles, between LDH activity in the liver of foothill and lowland populations of frogs suggests that the sensitivity of liver LDH to changes in oxygen tension is higher. The vector of a number of other kinetic parameters of LDH (Ki, Sopt, Δ) in the liver of animals from mountainous landscapes is absolutely opposite to that of skeletal muscles. High activity and modifications of the catalytic properties of LDH in the tissues of marsh frogs living in mid-mountain areas may play an important role in the adaptation of these animals to conditions of oxygen deficiency.

Metabolomic Profiling Reveals Differences in Hypoxia Response between Far Eastern and Siberian Frogs.

Anoxia is a significant challenge for most animals, as it can lead to tissue damage and death. Among amphibians, the Siberian frog Rana amurensis is the only known species capable of surviving near-zero levels of oxygen in water for a prolonged period. In this study, we aimed to compare metabolomic profiles of the liver, brain, and heart of the Siberian frog exposed to long-term oxygen deprivation (approximately 0.2 mg/L water) with those of the susceptible Far Eastern frog (Rana dybowskii) subjected to short-term hypoxia to the limits of its tolerance. One of the most pronounced features was that the organs of the Far Eastern frog contained more lactate than those of the Siberian frog despite a much shorter exposure time. The amounts of succinate were similar between the two species. Interestingly, glycerol and 2,3-butanediol were found to be significantly accumulated under hypoxia in the Siberian frog, but not in the Far Eastern frog. The role and biosynthesis of these substances are still unclear, but they are most likely formed in certain side pathways of glycolysis. Based on the obtained data, we suggest a pathway for metabolic changes in the Siberian frog under anoxia.

Complete genome sequence of Candidatus Mycobacterium wuenschmannii, a nontuberculous mycobacterium isolated from a captive population of Amazon milk frogs.

A slow growing species of nontuberculous mycobacteria (NTM) was isolated from the liver of an Amazon milk frog. The complete genome of this isolate comprises 5,102,433 bp, exhibiting 66.86% GC content, 4,940 protein-coding sequences, 52 predicted RNA genes, and 39 repeat regions.

Characteristics of the interferon regulatory factor IRF5 and its stimulation expression by Elizabethkingia miricola Infection in black-spotted frog (Pelophylax nigromaculatus)

Interferon regulatory factors (IRFs) have been confirmed as vital transcription factors for the immune system, which play a certain role in anti-infection defense, immune regulation, hematopoietic cell development, and maturation of the immune system. In this study, the cDNA of Interferon regulatory factor 5 (IRF5) was cloned from black-spotted frog, Pelophylax nigromaculatus, and termed PnIRF5. The results indicated, the full-length cDNA of PnIRF5 was 2090 bp, and a putative protein of 504 amino acids was encoded. The results of protein domain prediction suggested that PnIRF5 exhibited a DNA-binding domain (DBD), a middle region (MR), an IRF association domain (IAD), a virus activated domain (VAD), as well as two nuclear localization signals (NLSs). Phylogenetic analysis clustered PnIRF5 into the amphibian IRF5 subgroup in vertebrate IRF5 group. The results of qPCR show that PnIRF5 mRNA was expressed in all examined tissues, with higher levels were identified in the kidney, intestine, and lung. The gene expression of PnIRF5 was analyzed in kidney, spleen, and liver of black-spotted frog after challenged with Elizabethkingia miricola, and the maximum expression level of PnIRF5 in liver, spleen, and kidney were 1.80, 2.33 and 2.88 times that at 0 days post challenged, respectively. As indicated by the results, the PnIRF5 protein may take on critical significance in protecting black-spotted frog from E. miricola infection, and they may play a certain role in the immune response of black-spotted frog against bacteria.

Hepatic citrate synthase suppression in the freeze-tolerant wood frog (Rana sylvatica)

The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the Vmax of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters.

Freeze-induced suppression of pyruvate kinase in liver of the wood frog (Rana sylvatica).

The wood frog (Rana sylvatica) undergoes physiological and metabolic changes to withstand subzero temperatures and whole body freezing during the winter months. Along with metabolic rate depression, high concentrations of glucose are produced as a cryoprotectant by liver and distributed to all other tissues. Pyruvate kinase (PK; EC:2.7.1.40), the final enzyme of glycolysis, plays an important role in the modulation of glucose metabolism and, therefore, overall metabolic regulation. The present study investigated the functional and kinetic properties of purified PK from liver of control (5°C acclimated) and frozen (-2.5°C for 24h) wood frogs. Liver PK was purified to homogeneity by a two-step chromatographic process, followed by analysis of enzyme properties. A significant decrease in the affinity of PK for its substrates, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) at 22°C and 5°C was noted in liver from frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant increase in serine and threonine phosphorylation by 1.46-fold and 1.73- fold for PK from frozen frogs as compared with controls. Furthermore, a test of thermal stability showed that PK from liver of frozen wood frogs showed greater stability as compared with PK from control animals. Taken together, these results suggest that PK is negatively regulated, and glycolysis is suppressed, during freezing. This response acts as an important survival strategy for maintaining continuously elevated levels of cryoprotectant in frogs while they remain in a hypometabolic frozen state.

Exposure to ionizing radiation and liver histopathology in the tree frogs of Chornobyl (Ukraine)

Ionizing radiation has the potential to damage organic molecules and decrease the health and survival of wildlife. The accident at the Chornobyl Nuclear Plant (Ukraine, 1986) led to the largest release of radioactive material to the environment. Among the different organs of a vertebrate, the liver plays a crucial role in detoxification processes, and has been used as a biomarker to investigate cellular damage in ecotoxicological research. Here, we examined the impact of the exposure to the current levels of ionizing radiation present in the Chornobyl Exclusion Zone on the liver of Eastern tree frogs (Hyla orientalis). We quantified the area of melanomacrophage cells and morphological variables of hepatocytes, two cell types often used to estimate damage caused by pollutants in vertebrates. First, we investigated whether these hepatic parameters were indicative of frog (individual) condition. Then, we analyzed the effect of individual absorbed dose rates and ambient radiation levels on frog livers. Most of the studied parameters were correlated with individual body condition (a good predictor of amphibian fitness and survival). We did not detect marked morphological lesions in the liver of frogs captured in medium-high radiation environments. The area occupied by melanomacrophages and the morphology of hepatocytes did not change across a gradient of radiocontamination covering two orders of magnitude. Once accounting for body condition and sampling locality, the area of melanomacrophages was lower in areas with high radiation levels. Finally, the area occupied by melanomacrophages was not linked to dorsal skin coloration. Our results indicate that current levels of radiation experienced by tree frogs in Chornobyl do not cause histopathological damage in their liver. These results agree with previous physiological work in the species in the Chornobyl area, and encourage further molecular and physiological research to fully disentangle the current impact of the Chornobyl accident on wildlife.

Isolation of bacteria through gram staining from liver of Frog

Isolation of bacteria through gram staining from liver of Frog

One step purification and regulation of fructose 1,6-bisphosphatase from liver of the freeze tolerant wood frog, Rana sylvatica

One step purification and regulation of fructose 1,6-bisphosphatase from liver of the freeze tolerant wood frog, Rana sylvatica

Role of unfolded protein response and ER-associated degradation under freezing, anoxia, and dehydration stresses in the freeze-tolerant wood frogs.

The North American amphibian, wood frogs, Rana sylvatica are the most studied anuran to comprehend vertebrate freeze tolerance. Multiple adaptations support their survival in frigid temperatures during winters, particularly their ability to produce glucose as natural cryoprotectant. Freezing and its component consequences (anoxia and dehydration) induce multiple stresses on cells. Among these is endoplasmic reticulum (ER) stress, a condition spawned by buildup of unfolded or misfolded proteins in the ER. The ER stress causes the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway that potentially could lead to apoptosis. Immunoblotting was used to assess the responses of major proteins of the UPR and ERAD under freezing, anoxia, and dehydration stresses in the liver and skeletal muscle of the wood frogs. Targets analyzed included activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and EDEM (ERAD enhancing α-mannosidase-like proteins) and XBP1 (X-box binding protein 1) proteins. UPR signaling was triggered under all three stresses (freezing, anoxia, dehydration) in liver and skeletal muscle of wood frogs with most tissue/stress responses consistent with an upregulation of the primary targets of all three UPR pathways (ATF4, ATF6, and XBP-1) to enhance the protein folding/refolding capacity under these stress conditions. Only frozen muscle showed preference for proteasomal degradation of misfolded proteins via upregulation of EDEM (ERAD). The ERAD response of liver was downregulated across three stresses suggesting preference for more refolding of misfolded/unfolded proteins. Overall, we conclude that wood frog organs activate the UPR as a means of stabilizing and repairing cellular proteins to best survive freezing exposures.

Tunable Toxicity of Bufadienolides is Regulated through a Configuration Inversion Catalyzed by a Short-Chain Dehydrogenase/Reductase.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14β-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(β→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

Status of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in liver and skin of the freeze tolerant wood frog

The wood frog (Rana sylvatica) has adapted full-body freezing and thawing as a means of sub-zero winter survival and early-breeding in ephemeral pools. One such protective process implicated recently in freeze-thaw tolerance is that of anti-apoptotic signaling, which has been proposed to play a cytoprotective role by modulating stress-induced death signals. This study employed the use of immunoblotting to examine response of a potent cell cycle and apoptosis regulator, known as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, to freezing and thawing in the liver and skin of the wood frog. This pathway demonstrably exhibits factor- and tissue-specific changes between non-frozen, 24 h-frozen, and 8 h-thawed conditions. There were few changes in JAK-STAT proteins in frozen frogs, but protective changes were observed upon thaw: Elevated levels of pJAK3 and nuclear localization of pSTAT3 and pSTAT5 suggested an increase in anti-apoptotic signaling after thaw. By contrast, both STAT1 and STAT3 signaling appeared to increase in frozen skin, suggesting frogs use homeostatic regulation of apoptotic- and anti-apoptotic signals, in an antagonistic and compensatory manner. As such, these findings support that JAK-STAT pathway signaling modulation is a plausible adaptation that contributes to fast and reversible manipulation of anti-apoptotic signals, thus assisting in freeze survival of the wood frog.

Per- and Polyfluoroalkyl Substances (PFASs) Impair Lipid Metabolism in Rana nigromaculata: A Field Investigation and Laboratory Study.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous environmental pollutants, causing environmental threats and public health concerns, but information regarding PFAS hepatotoxicity remains elusive. We investigated the effects of PFASs on lipid metabolism in black-spotted frogs through a combined field and laboratory study. In a fluorochemical industrial area, PFASs seriously accumulate in frog tissues. PFAS levels in frog liver tissues are positively related to the hepatosomatic index along with triglyceride (TG) and cholesterol (TC) contents. In the laboratory, frogs were exposed to 1 and 10 μg/L PFASs, respectively (including PFOA, PFOS, and 6:2 Cl-PFESA). At 10 μg/L, PFASs change the hepatic fatty acid composition and significantly increase the hepatic TG content by 1.33 to 1.87 times. PFASs induce cross-talk accumulation of TG, TC, and their metabolites between the liver and serum. PFASs can bind to LXRα and PPARα proteins, further upregulate downstream lipogenesis-related gene expression, and downregulate lipolysis-related gene expression. Furthermore, lipid accumulation induced by PFASs is alleviated by PPARα and LXRα antagonists, suggesting the vital role of PPARα and LXRα in PFAS-induced lipid metabolism disorders. This work first reveals the disruption of PFASs on hepatic lipid homeostasis and provides novel insights into the occurrence and environmental risk of PFASs in amphibians.