When the brain goes diving: transcriptome analysis reveals a reduced aerobic energy metabolism and increased stress proteins in the seal brain.

BACKGROUND: During long dives, the brain of whales and seals experiences a reduced supply of oxygen (hypoxia). The brain neurons of the hooded seal (Cystophora cristata) are more tolerant towards low-oxygen conditions than those of mice, and also better survive other hypoxia-related stress conditions like a reduction in glucose supply and high concentrations of lactate. Little is known about the molecular mechanisms that support the hypoxia tolerance of the diving brain. RESULTS: Here we employed RNA-seq to approach the molecular basis of the unusual stress tolerance of the seal brain. An Illumina-generated transcriptome of the visual cortex of the hooded seal was compared with that of the ferret (Mustela putorius furo), which served as a terrestrial relative. Gene ontology analyses showed a significant enrichment of transcripts related to translation and aerobic energy production in the ferret but not in the seal brain. Clusterin, an extracellular chaperone, is the most highly expressed gene in the seal brain and fourfold higher than in the ferret or any other mammalian brain transcriptome. The largest difference was found for S100B, a calcium-binding stress protein with pleiotropic function, which was 38-fold enriched in the seal brain. Notably, significant enrichment of S100B mRNA was also found in the transcriptomes of whale brains, but not in the brains of terrestrial mammals. CONCLUSION: Comparative transcriptomics indicates a lower aerobic capacity of the seal brain, which may be interpreted as a general energy saving strategy. Elevated expression of stress-related genes, such as clusterin and S100B, possibly contributes to the remarkable hypoxia tolerance of the brain of the hooded seal. Moreover, high levels of S100B that possibly protect the brain appear to be the result of the convergent adaptation of diving mammals.

Seasonal variations in the intermediate metabolism in South American tree-frog Boana pulchella.

Seasonal metabolic changes can be observed in many anurans' species. In subtropical environments with environmental temperatures variations, the temperature is a factor that can influence the extent and intensity of activity in many anuran species. Nonetheless, some species of subtropical frogs may remain active throughout the year. Boana pulchella, a subtropical species, seems to be able to survive low temperatures and remain reproductively active even in the coldest months. Therefore, we hypothesized that B. pulchella presents seasonal changes in the energy metabolism to sustain activity during all year. This study evaluated the main energy substrate levels and metabolism of B. pulchella in plasma, liver and muscle of male individuals collected in winter, spring, summer and fall in the state of Rio Grande do Sul, Brazil. Our results showed that B. pulchella has a higher glycolytic oxidation rate in liver (P = 0.0152) and muscle (P = 0.0003) and higher glycogenesis from glucose in muscle (P = 0.0002) in summer, indicating the main energy substrates in this season is glucose. The higher muscle glycogen (P = 0.0008) and lower plasma glucose in fall (P = 0.0134) may indicate an anticipatory regulation for storing to the most thermally demanding cold period: winter. These results indicated seasonal differences in the main energy substrates, and these metabolic changes among seasons can be part of a metabolic adjustment allowing maintenance of reproductive activity all year in Boana pulchella species.

Effects of tributyltin (TBT) on the intermediate metabolism of the crab Callinectes sapidus.

This study investigated if the exposure to tributyltin (TBT), a chemical used worldwide in boat antifouling paints, could result in metabolic disturbances in the blue crab Callinectes sapidus. After the exposure to TBT 100 or 1000 ng.L-1 for 48 and 96 h, hemolymph and tissues were collected to determine the concentration of metabolites and lipid peroxidation. The levels of glucose, lactate, cholesterol, and triglycerides in the hemolymph were not affected by TBT exposure. Hemolymph protein and heart glycogen increased in the crabs exposed to TBT 1000 for 96 h. Anterior gills protein and lipoperoxidation decreased after 96 h in all groups. These results suggest that C. sapidus can maintain energy homeostasis when challenged by the TBT exposure for 48 h and that metabolic alterations initiate after 96 h.

Lactate Administration Reduces Brain Injury and Ameliorates Behavioral Outcomes Following Neonatal Hypoxia-Ischemia.

Neonatal hypoxic-ischemic encephalopathy is a major cause of mortality and disability in newborns and the only standard approach for treating this condition is therapeutic hypothermia, which shows some limitations. Thus, putative neuroprotective agents have been tested in animal models. The present study evaluated the administration of lactate, a potential energy substrate of the central nervous system (CNS) in an animal model of hypoxia-ischemia (HI), that mimics in neonatal rats the brain damage observed in human newborns. Seven-day-old (P7) male and female Wistar rats underwent permanent common right carotid occlusion combined with an exposition to a hypoxic atmosphere (8% oxygen) for 60 min. Animals were assigned to four experimental groups: HI, HI + LAC, SHAM, SHAM + LAC. Lactate was administered intraperitoneally 30 min and 2 h after hypoxia in HI + LAC and SHAM + LAC groups. HI and SHAM groups received vehicle at the same time points. The volume of brain lesion was evaluated in P9. Animals underwent behavioral assessments: negative geotaxis, righting reflex (P8 and P14), and cylinder test (P20). Lactate administration reduced the volume of brain lesion and improved behavioral parameters after HI in both sexes. Thus, lactate administration could be a neuroprotective strategy for the treatment of neonatal HI, a disorder still affecting a significant percentage of human newborns.

Evaluation of the effects of vitamin A supplementation on adult rat substantia nigra and striatum redox and bioenergetic states: mitochondrial impairment, increased 3-nitrotyrosine and alpha-synuclein, but decreased D2 receptor contents.

Vitamin A at moderate to high doses is applied in the treatment of some life threatening pathological conditions, for instance cancers. Additionally, vitamin A at low concentrations is a known antioxidant molecule. However, by increasing vitamin A (or its derivatives) concentrations, there is an increase in the levels of oxidative stress markers in several experimental models. Furthermore, it was reported that vitamin A therapy at high doses might induce cognitive decline among the patients, which may become anxious or depressive, for example, depending on vitamin A levels intake. We have previously reported increased levels of oxidative stress markers in rat substantia nigra and striatum. However, the mechanism by which this vitamin altered the redox environment in such rat brain regions remains to be elucidated. In the herein presented work, we have investigated the effects of vitamin A supplementation at clinical doses (1000-9000 IU/kg day(-1)) for 28 days on rat substantia nigra and striatum mitochondrial electron transfer chain (METC) activity, which may produce superoxide anion radical (O(2)(-*)) when impaired. Additionally, the levels of non-enzymatic antioxidant defenses were evaluated, as well as 3-nitrotyrosine, alpha- and beta-synucleins and TNF-alpha levels through ELISA assay. We observed impaired METC in both rat brain regions. Moreover, we found increased O(2)(-*) production and nitrotyrosine content in the nigrostriatal axis of vitamin A-treated rats, suggesting that the use of vitamin A at therapeutic doses may be rethought due to this toxic effects found here.

Characterization of different DNA repair pathways in hepatic cells of Zebrafish (Danio rerio).

The concern about DNA damage has directed efforts toward evaluating the genotoxic potential of physical and chemical agents. Since the extent of DNA damage is also related to the capacity of the organism in repairing the DNA, the advance of toxicological studies on this area depends on the characterization of the DNA repair mechanisms in the available models. The cellular zebrafish models, for example, replace mammalian cells to answer ecologically relevant questions on aquatic toxicology. So, the aim of the present study was to characterize the nucleotide excision repair (NER) and photoreactivation (PER) in two cellular models of Danio rerio liver, primary hepatocytes and ZF-L (Zebrafish Liver) cell line. We performed kinetic studies of the DNA damage levels after exposure to 6.8 J/m2 UVC using the T4-PDG modified Comet Assay, and determined the expression levels of important genes involved in NER, PER and base excision repair using RT-qPCR. It was observed that both ZF-L cell line and primary hepatocytes exhibit similar NER and PER activity. Primary hepatocytes showed similarities in the gene expression of most of the evaluated repair genes with the original tissue. These results indicate that both primary hepatocytes and ZF-L cells are useful models for toxicological studies aiming to evaluate NER and PER in hepatic cells. Moreover, the similarities in gene expression between the cellular models suggest that the ZF-L cells retain the DNA repair characteristics of the primary hepatocytes and, thus, could serve as replacement to this primary culture, reducing the use of animals in research.

Transcriptome Analysis Identifies Key Metabolic Changes in the Hooded Seal (Cystophora cristata) Brain in Response to Hypoxia and Reoxygenation.

The brain of diving mammals tolerates low oxygen conditions better than the brain of most terrestrial mammals. Previously, it has been demonstrated that the neurons in brain slices of the hooded seal (Cystophora cristata) withstand hypoxia longer than those of mouse, and also tolerate reduced glucose supply and high lactate concentrations. This tolerance appears to be accompanied by a shift in the oxidative energy metabolism to the astrocytes in the seal while in terrestrial mammals the aerobic energy production mainly takes place in neurons. Here, we used RNA-Seq to compare the effect of hypoxia and reoxygenation in vitro on brain slices from the visual cortex of hooded seals. We saw no general reduction of gene expression, suggesting that the response to hypoxia and reoxygenation is an actively regulated process. The treatments caused the preferential upregulation of genes related to inflammation, as found before e.g. in stroke studies using mammalian models. Gene ontology and KEGG pathway analyses showed a downregulation of genes involved in ion transport and other neuronal processes, indicative for a neuronal shutdown in response to a shortage of O2 supply. These differences may be interpreted in terms of an energy saving strategy in the seal's brain. We specifically analyzed the regulation of genes involved in energy metabolism. Hypoxia and reoxygenation caused a similar response, with upregulation of genes involved in glucose metabolism and downregulation of the components of the pyruvate dehydrogenase complex. We also observed upregulation of the monocarboxylate transporter Mct4, suggesting increased lactate efflux. Together, these data indicate that the seal brain responds to the hypoxic challenge by a relative increase in the anaerobic energy metabolism.

An atypical distribution of lactate dehydrogenase isoenzymes in the hooded seal (Cystophora cristata) brain may reflect a biochemical adaptation to diving.

The brains of some diving mammals can withstand periods of severe hypoxia without signs of deleterious effects. This may in part be due to an enhanced cerebral capacity for anaerobic energy production. Here, we have tested this hypothesis by comparing various parameters of the lactate dehydrogenase (LDH) in the brain of the hooded seal (Cystophora cristata) with those in the brains of the ferret (Mustela putorius furo) and mouse (Mus musculus). We found that mRNA and protein expression of lactate dehydrogenase a (LDHA) and lactate dehydrogenase b (LDHB), and also the LDH activity were significantly higher in the ferret brain than in brains of the hooded seal and the mouse (p < 0.0001). No conspicuous differences in the LDHA and LDHB sequences were observed. There was also no difference in the buffering capacities of the brains. Thus, an enhanced capacity for anaerobic energy production likely does not explain the higher hypoxia tolerance of the seal brain. However, the brain of the hooded seal had higher relative levels of LDHB isoenzymes (LDH1 and LDH2) compared to the non-diving mammals. Moreover, immunofluorescence studies showed more pronounced co-localization of LDHB and glial fibrillary acidic protein in the cortex of the hooded seal. Since LDHB isoenzymes primarily catalyze the conversion of lactate to pyruvate, this finding suggests that the contribution of astrocytes to the brain aerobic metabolism is higher in the hooded seal than in non-diving species. The cerebral tolerance of the hooded seal to hypoxia may therefore partly rely on different LDH isoenzymes distribution.

Oxidative stress and DNA damage responses to phenanthrene exposure in the estuarine guppy Poecilia vivipara.

Despite ubiquitous phenanthrene contamination in aquatic coastal areas, little is known regarding its potential effects on estuarine fishes. The present work evaluated the response of a large suite of oxidative stress- and DNA damage-related biomarkers to phenanthrene exposure (10, 20 and 200 µg L(-1), 96 h) using DMSO as the solvent in estuarine guppy Poecilia vivipara (salinity 24 psu). Phenanthrene affected oxidative stress-related parameters, and decreased antioxidant defenses and reactive oxygen species in the gills and muscle overall. Lipid peroxidation occurred in muscle at 200 µg L(-1) phenanthrene. Genotoxicity was increased at 20 µg L(-1), while 200 µg L(-1) caused a relative decrease in erythrocyte release into the bloodstream. These findings indicated that phenanthrene is genotoxic and can induce oxidative stress, depending on tissue and phenanthrene concentration analyzed. Thus, some of the biomarkers analyzed in the present study are sufficiently sensitive to monitor the exposure of the guppy P. vivipara to phenanthrene in salt water. However, further studies are required for a better interpretation of the dose-response patterns observed.

Biomarkers of waterborne copper exposure in the guppy Poecilia vivipara acclimated to salt water.

The responses of a large suite of biochemical and genetic parameters were evaluated in tissues (liver, gills, muscle and erythrocytes) of the estuarine guppy Poecilia vivipara exposed to waterborne copper in salt water (salinity 24 ppt). Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase, and glutathione S-transferase), metallothionein-like protein concentration, reactive oxygen species (ROS) content, antioxidant capacity against peroxyl radicals (ACAP), and lipid peroxidation (LPO) were evaluated in liver, gills, and muscle. Comet assay score and nuclear abnormalities and micronucleated cell frequency were analyzed in peripheral erythrocytes. The responses of these parameters were evaluated in fish exposed (96 h) to environmentally relevant copper concentrations (5, 9 and 20 µg L⁻¹). In control and copper-exposed fish, no mortality was observed over the experimental period. Almost all biochemical and genetic parameters proved to be affected by waterborne copper exposure. However, the response of catalase activity in liver, ROS, ACAP and LPO in muscle, gills and liver, and DNA damages in erythrocytes clearly showed to be dependent on copper concentration in salt water. Therefore, the use of these parameters could be of relevance in the scope of biomonitoring programs in salt water environments contaminated with copper.

Decreased anxiety-like behavior and locomotor/exploratory activity, and modulation in hypothalamus, hippocampus, and frontal cortex redox profile in sexually receptive female rats after short-term exposure to male chemical cues.

Chemical cues are widely used for intraspecific social communication in a vast majority of living organisms ranging from bacteria to mammals. As an example, mammals release olfactory cues with urine that promote neuroendocrine modulations with changes in behavior and physiology in the receiver. In this work, four-month-old Wistar (regular 4-day cyclic) virgin female rats were utilized in the proestrus-to-estrus phase of the reproductive cycle for experimental exposure. In an isolated room, female rats were exposed for 90 min to male-soiled bedding (MSB). Elevated plus-maze assay, open field test, and light/dark box task were performed to analyze behavioral alterations on females after exposure. For biochemical assays, female rats were killed and the hypothalamus, hippocampus, and frontal cortex were isolated for further analysis. Antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase), non-enzymatic antioxidant defense measurements (TRAP and TAR), and the oxidative damage parameters (TBARS, Carbonyl and SH content) were analyzed. In behavioral analyses we observe that female rats show decreased anxiety and locomotory/exploratory activities after MSB exposure. In biochemical assays we observed an increase in both enzymatic and non-enzymatic antioxidant defenses in different central nervous system (CNS) structures analyzed 30 and 90 min after MSB exposure. Furthermore, hippocampus and frontal cortex showed diminished free radical oxidative damage at 180 and 240 min after exposure. These results provide the first evidence that oxidative profile of female CNS structures are altered by chemical cues present in the MSB, thus suggesting that pheromonal communication is able to modulate radical oxygen species production and/or clearance in the female brain.

Impacto do triatlon ironman sobre os parametros de estresse oxidativo / Impact of ironman triathlon on oxidative stress parameters

Vários estudos têm investigado as alterações na resposta bioquímica em triatletas participantes de provas de Ironman, mas poucos dados relatam as mudanças de estresse oxidativo. O estudo teve como objetivo investigar os parâmetros de estresse oxidativo em triatletas após corrida de Ironman. Participaram do estudo, dezoito triatletas do sexo masculino, com idade média de 34,5 ± 2,15 anos, peso 69,3 ± 1,9 kg e altura 1,71 ± 0,18 m participaram do estudo. A corrida de Ironman consiste em 3,8 km de natação, 180 km de bicicleta e 42,2 km de corrida. Antes da corrida e imediatamente após seu término foi retirado 10 mL de sangue, sendo o mesmo centrifugado e armazenado o soro em freezer -80ºC para posteriores análises. A capacidade antioxidante total, lipoperoxidação, carbonilação de proteínas e conteúdo total de tióis foram determinadas. Os resultados mostraram um aumento significativo na quantidade de hidroperóxidos, carbonilação de proteínas e uma redução na capacidade antioxidante total do plasma e no conteúdo total de tióis após a prova (p<0.05) em relação à pré-prova, concluindo que a prova de Ironman provoca alterações significativas nos marcadores de estresse oxidativo em atletas e que uma suplementação com antioxidantes seria importante para reverter estes efeitos.

Several studies have investigated the biochemical response changes that take place in Ironman triathletes, but there are few data on oxidative stress changes. The objective of this study was to investigate oxidative stress parameters in triathletes after an Ironman event. The sample consisted of eighteen male triathletes, with a mean age of 34.5 ± 2.15 years, weight 69.3 ± 1.9 kg, and height 1.71 ± 0.18 m. The Ironman triathlon consists of a 3.8-km swim, a 180-km bicycle ride, and a 42.2-km (marathon) run. Before the competition and immediately after its conclusion, 10-mL blood samples were collected, centrifuged and frozen at -80ºC for subsequent analysis. Total antioxidant capacity, lipid peroxidation, protein carbonylation, and total thiol content were measured. The results showed a significant increase in all markers after the event (p<0.05) in relation to the pre-event period, which conclusively shows that the Ironman triathlon induces significant changes oxidative stress markers in athletes and that antioxidant supplementation would be important to reverse these effects.