Physiological strategies of acute thermal conditions of Rhamdia voulezi collected in the Iguaçu river watershed, Paraná, Brazil: biochemical markers of metabolic and oxidative stress.

Thermal pollution creates substantial challenges that alter energy demand and produce reactive oxygen species that damage fish DNA, proteins, and lipids. Rhamdia voulezi is a species of fish native to the Iguaçu river, Paraná, Brazil, that does not have scientific records of minimum (CTmin) and maximum (CTmax) temperatures required for survival. As it is a top predator species in the food chain and lives at temperatures below 22 °C, the loss of the species can cause functional problems in controlling the ecosystem and energy flow. The study evaluated the tissue metabolism of the brain, heart, and muscle of R. voulezi (n = 72) subjected to acute thermal stress of 31 °C for 2, 6, 12, 24, and 96 h after acclimatization to 21 °C. The biochemical markers SOD, GPx, MDH, HK, and CK of the brain, PCO of the heart and CAT, glycogen, G6PDH, and ALT of muscle were significant. PCA, IBR, thermal sensitive, and condition factor suggested that R. voulezi has different physiological strategies for acclimatization to 31 °C to mobilize and sustain the metabolic needs of oxygenation and energy allocation/utilization for tissue ATP production.

Assessing physiological responses and oxidative stress effects in Rhamdia voulezi exposed to high temperatures.

Exposure to high temperatures induces changes in fish respiration, resulting in an increased production of reactive oxygen species. This, in turn, affects the enzymatic and non-enzymatic components of antioxidant defenses, which are essential for mitigating cellular stress. Rhamdia voulezi, an economically important fish species endemic to Brazil's Iguaçu River, served as the subject of our study. Our goal was to assess enzymatic antioxidant biomarkers (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glucose-6-phosphate dehydrogenase), non-protein thiol levels (reduced glutathione), and markers of oxidative damage (lipoperoxidation and carbonylation) in the liver, gills, and kidneys of R. voulezi after acute exposure to high temperatures (31°C) for 2, 6, 12, 24, and 96 h. Control groups were maintained at 21°C. Our findings revealed that the liver exhibited increased superoxide dismutase levels up to 12 h and elevated glutathione S-transferase levels at 12 and 96 h at 31°C. In the gills, superoxide dismutase levels increased up to 24 h, along with increased lipoperoxidation at 2, 6, 12, and 96 h of exposure to high temperatures. The kidneys responded to heat stress at 12 h, with an increase in superoxide dismutase and catalase activity, and lipid peroxidation was observed at 2 and 6 h at 31°C. The three tissues evaluated responded differently to heat stress, with the liver demonstrating greater physiological adjustment to high temperatures. The intricate interplay of various antioxidant defense biomarkers and oxidative damage suggests the presence of oxidative stress in R. voulezi when exposed to high temperatures.

Metabolic responses in the gills of Yellowtail Lambari Astyanax lacustris under low- and high-temperature thermal stress.

OBJECTIVE: Ectothermic fish are directly affected by temperature changes in the environment. The aim of this study was to evaluate the metabolic responses in the gills of Yellowtail Lambari Astyanax lacustris under thermal stress. METHODS: To this end, we used spectrophotometry to evaluate the biomarkers of carbohydrate and protein metabolism, antioxidant defense, and oxidative damage in fish subjected to low (15°C) and high (31°C) temperatures, with control groups held at 23°C, for 2, 6, 12, 24, 48, and 96 h. RESULT: The results showed that cold thermal stress did not change the energy demand, and the antioxidant defense was reduced; therefore, the gills were vulnerable to the action of reactive oxygen species (ROS), presenting increased protein carbonylation at 12 h. With heat thermal stress, a higher energy demand was observed, which was verified by an increase in aerobic metabolism by glycolysis and the citric acid cycle. High-temperature stress also increased the antioxidant defenses, as verified by the increased activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase. However, the antioxidant defense system could not protect tissues from the action of ROS, as protein carbonylation increased at 6 and 24 h, indicating oxidative stress. CONCLUSION: The results showed that (1) temperature variations caused metabolic adjustments in the gills of Yellowtail Lambari, (2) the adaptive responses were different for winter and summer temperatures, and (3) Yellowtail Lambari recovered homeostasis when subjected to thermal stress, even with the occurrence of oxidative stress.

Integrated biomarker response index as an ally in the observation of metabolic biomarkers in muscle of Astyanax lacustris exposed to thermal variation.

Temperature is an important factor that conditions the physiological responses of fish, generating a stressful condition when in non-ideal parameters. Thus, the objective was to evaluate metabolic aspects in the muscle of fish Astyanax lacustris submitted to thermal shock. The specimens were subjected to 2, 6, 12, 24, 48, and 96 h of exposure to 15 °C and 31 1 °C, with their respective controls 23 °C. At 15 °C there was a reduction in glycogenolysis in the initial periods indicated by changes in glycogen phosphorylase (GP), pyruvate and lactate. Subsequently, there was an increase in GP activity, pyruvate levels and hexokinase activity in the next time periods, suggesting an increase in energy demand. At 31 °C there was observed low need for the protein metabolism, indicated by reduction in the activity of aspartate aminotransferase and alanine aminotransferase. At 15 °C, initial periods show an increase in glutathione reductase activity and an increase in carbonylated protein levels, indicating induction of oxidative stress for muscle. At 31 °C, there was a punctual increase in reduced glutathione levels at 24 h. In addition, the integrated biomarker response index proved to be a good ally in the evaluation of a set of biomarkers, corroborating the results observed by the biomarkers individually. Thus, it is possible to conclude that the acute thermal shock affects the metabolism of A. lacustris muscle, which undergoes rearrangements to deal with temperature, where 15 °C is more stressful than 31 °C.

Can high- and low-temperature thermal stress modulate the antioxidant defense response of Astyanax lacustris brain?

Change in temperature of aquatic environment have impacts on the physiology of fish, especially in the brain, which is a vital organ and prone to oxidative damage. Astyanax lacustris is a freshwater fish that play an important role in the food market and has been increasingly used in fish farms, besides environmental monitoring studies. Therefore, this study aimed to evaluate the responses of antioxidant biomarkers and products of the oxidative process in the brains A. lacustris subjected to thermal shock. The specimens were obtained from artificial farming lakes and subjected to shock induced by exposure to high (31 °C ± 0.5) and low (15 °C ± 0.5) temperature for 2, 6, 12, 24, 48, 72 and 96 h; control group were maintained at 23 °C ± 0.5. At 31 °C, glutathione-related enzymes were more responsive, suggested by the change activity of GPx and G6PDH enzymes, in addition to GSH levels. At 15 °C, enzymes of the first line of defense were more active, evidenced by the change CAT activity. No significant changes were detected in the levels of ROS, LPO and PCO. These results indicate that the brains of A. lacustris have an efficient antioxidant defense system with the ability to acclimatize to the temperatures tested.

Effects of heat shock on energy metabolism and antioxidant defence in a tropical fish species Psalidodon bifasciatus.

Predictions about global warming have raised interest in assessing whether ectothermic organisms will be able to adapt to these changes. Understanding the physiological mechanisms and metabolic adjustment capacity of fish subjected to heat stress can provide subsidies that may contribute to decision-making in relation to ecosystems and organisms subjected to global climate change. This study investigated the antioxidant defence system and energy metabolism of carbohydrate and protein responses in the gill, liver and kidney tissues of Psalidodon bifasciatus (Garavello & Sampaio 2010), a Brazilian freshwater fish used in aquaculture and in biological studies, following exposure to heat shock at 31°C for 2, 6, 12, 24 and 48 h. The fish presented signs of stress in all tissues tested, as evidenced by increased lipid peroxidation concentration at 2 h and phosphofructokinase, hexokinase and malate dehydrogenase activity at 48 h in the gills; increased glutathione-S-transferase activity at 12 h, citrate synthase activity at 24 h and concentration of reduced glutathione (GSH) concentration at 12 and 48 h in the liver; and through increased activity of superoxide dismutase at 48 h, glutathione reductase at 24 h, glucose-6-phosphate dehydrogenase at 48 h and concentration of GSH at 24 h in the kidney. In the kidneys, changes in the antioxidant system were more prominent, whereas in the gills, there were greater changes in the carbohydrate metabolism. These results indicated the importance of glycolysis and aerobic metabolism in the gills, aerobic metabolism in the liver and pentose-phosphate pathway in the kidneys during homeostasis. The biomarker response was tissue specific, with the greatest number of biomarkers altered in the gills, followed by those in the kidneys and liver.

Gradual increase of temperature trigger metabolic and oxidative responses in plasma and body tissues in the Antarctic fish Notothenia rossii.

Antarctica is considered a thermally stable ecosystem; however, climate studies point to increases in water temperatures in this region. These thermal changes may affect the biological processes and promote metabolic changes in the adapted organisms that live in this region, rendering the animals more vulnerable to oxidative damage. This study assessed the effect of acclimation temperature on the levels of stress response markers in plasma, kidney, gill, liver, and brain tissues of Notothenia rossii subjected to gradual temperature changes of 0.5 °C/day until reaching temperatures of 2, 4, 6, and 8 °C. Under the effect of the 0.5 °C/day acclimation rate, gill tissue showed increased glutathione-S-transferase (GST) activity; kidney tissue showed increased H+-ATPase activity. In the liver, there was also an increase in GSH. In plasma, gradual decreases in the concentrations of total proteins and globulins were observed. These responses indicate a higher production of reactive oxygen species ROS, an imbalance in energy demand, and a lack in protein synthesis. Gradual increase in temperature may cause opposite responses to the thermal shock model in N. rossii.

Cold and warm waters: energy metabolism and antioxidant defenses of the freshwater fish Astyanax lacustris (Characiformes: Characidae) under thermal stress.

Subtropical fish are exposed to seasonal variations in temperature that impose a set of adaptations on their metabolism necessary for the maintenance of homeostasis. In this study, we addressed the effects of temperature variation on the metabolism of Astyanax lacustris, a species of freshwater fish common in the subtropical region of Brazil. Biomarkers of carbohydrate and protein metabolism, antioxidant defense, and oxidative damage were evaluated in the liver of A. lacustris exposed to low (15 °C) and high (31 °C) temperature thermal shock, with controls at 23 °C for 2, 6, 12, 24, 48, 72, and 96 h. A high energy demand was observed during the first 48 h of exposure to 15 °C, which is necessary for metabolic adjustment at low temperatures, with an increase in glycolysis, citric acid cycle, and amino acid catabolism. In addition, at 31 °C, glucose was exported in the first 12 h of exposure, and an increase in the citric acid cycle suggested acetyl-CoA as the pathway substrate, originating from the oxidation of lipids. The antioxidant defenses did not change at 15 °C, as opposed to 31 °C, in which there were changes in several antioxidant defense markers, indicating a response to the production of ROS. However, oxidative stress was observed at both temperatures, with oxidative damage detected by lipid peroxidation at 15 °C and protein carbonylation at 31 °C.

Biomarkers of oxidative stress and cell damage in freshwater bivalves Diplodon parodizi exposed to landfill leachate.

Landfill is a public and environmental health problem; establishing and understanding methodologies to decrease its toxicity are thus necessary. Leachate samples were collected, at a sanitary landfill, immediately after the exit from the landfill, i.e. raw leachate (collection point A), after conventional treatment (point B) and after treatment by wetlands (point C). D. parodizi specimens were exposed to 3%, 10% and control (0%) dilutions of leachate from these collection points for 7 days. Markers of antioxidant defences and cell damage were analysed. At point B, the gills of D. parodizi showed higher glutathione-S-transferase (GST) and glutathione reductase (GR) activity; the latter is a supplier of glutathione reductase (GSH). The low GST activity at point A was associated with the hormesis effect. Higher levels of superoxide dismutase (SOD), ethoxyresorufin-O-deethylase (EROD) and glutathione peroxidase (GPx) occurred at point A. Glucose-6-phosphate dehydrogenase (G6PDH) was inhibited at the points with the highest pollutant load and at the highest leachate dilutions. Higher levels of markers at point A may be related to the high pollutant charge and specific compounds present in the untreated leachate. The multi-xenobiotic resistance mechanism (MXR), metallothionein-like proteins (MT) and lipid peroxidation (LPO) did not vary among treatments. The biomarker responses showed negative effects of the leachate on the freshwater bivalve and simultaneously showed that the wetland treatment employed at the Caximba sanitary landfill is effective.

Effect of gradual temperature increase on the carbohydrate energy metabolism responses of the Antarctic fish Notothenia rossii.

The warming of the Southern Ocean waters may affect the biological processes and the performance of the fish inhabiting it. The notothenioid group is metabolically specialized to low-temperature environments and may be vulnerable to the climatic changes imposed on the Antarctic continent. However, gradual temperature changes potentially allow an opportunity for plasticity adjustments. The present study evaluated the effect of gradual increase of temperature on the enzymatic and nonenzymatic parameters of energy metabolism in renal, branchial, hepatic, and encephalic tissue of Notothenia rossii subjected to a gradual temperature change of 0.5 °C/day until reaching 2 °C, 4 °C, 6 °C, and 8 °C. Under the effect of an acclimation rate of 0.5 °C/day, the gill tissue showed increased phosphofructokinase (PFK) enzyme activity. In the kidney, there was increased activity of the malate dehydrogenase (MDH), glucose-6-phosphatase (G6PDH), and glycogen phosphorylase (GP) enzymes. There was an increase in lactate concentration in the liver and an increase in GP enzyme activity in the brain. The specific tissue responses indicate the presence of thermal plasticity and an attempt to regulate energy metabolism to mitigate thermal stress in this species under these experimental conditions, possibly through the activation of glycolysis, gluconeogenesis, and glycogenolysis.

Metabolic responses in Antarctic Nototheniidae brains subjected to thermal stress.

Antarctic Nototheniidae is an attractive group for studying metabolic and physiological responses at high temperatures. The present work investigated the metabolic responses of the carbohydrate metabolism and antioxidant system to thermal stress at 8 °C (for 2-144 h) in the brains of Notothenia rossii and Notothenia coriiceps. In N. coriiceps, glycogenolysis was essential in the first hours of exposure (2 h) at 8 °C and, in addition to inhibiting glucose-6-phosphatase activity, was important for activating the pentose phosphate pathway. In N. rossii, anaerobic metabolism was reduced in the first hours of exposure (2 and 6 h) at 8 °C, followed by reduced hexokinase activity, suggesting energy regulation between neurons and astrocytes. The antioxidant system results indicated the importance of the actions of the glutathione-dependent antioxidant enzymes glutathione-S-transferase and glutathione peroxidase as well as those of catalase in N. coriiceps and the action of glutathione-S-transferase, glutathione peroxidase and glutathione reductase in N. rossii, especially during the first 12 h of thermal stress exposure. These results indicate tissue-specific patterns and species-specific responses to this stress.

Heat stress in the heart and muscle of the Antarctic fishes Notothenia rossii and Notothenia coriiceps: Carbohydrate metabolism and antioxidant defence.

Carbohydrate metabolism and the antioxidant defence system of heart and muscle of the Antarctic notothenioids Notothenia rossii and Notothenia coriiceps were evaluated in response to heat stress (8 °C) over 144 h. N. rossii heart exhibited decreased glycolysis and aerobic metabolism after up to 12 h of exposure to 8 °C, and anaerobiosis was inhibited within 24 h. However, these pathways were stimulated after 72 h at 8 °C. The consumption of glucose-6-phosphate, derived from hexokinase (HK), by glucose-6-phosphate dehydrogenase (G6PDH) decreased in N. rossii heart within 6 h at 8 °C, with a subsequent increase at 72 h. In N. rossii muscle at 8 °C, glycolysis was stimulated within 2 h by an increase in pyruvate kinase (PK), and aerobic metabolism was stimulated at 144 h, together with anaerobiosis. In N. coriiceps heart at 8 °C, glucose break down by HK decreased within 2 h and subsequently increased at 12 and 24 h. Increased glucose-6-phosphate consumption by G6PDH occurred within 6 h at 8 °C. In N. coriiceps muscle at 8 °C, glycolysis was stimulated at 2 and 6 h, with subsequent inhibition within 24 h, as indicated by HK activity. Aerobic metabolism was inhibited at 72 and 144 h at 8 °C through the inhibition of citrate synthase (CS). Heat stress caused responses were only occasional and transient in antioxidant defence system of both species in the heart and muscle, leading to increased glutathione (GSH) and decreased levels of lipoperoxidation in the heart of both species. The results obtained in this study in the heart and muscles indicate that under heat stress at 8 °C, N. rossii is more responsive than N. coriiceps with respect to carbohydrate metabolism.

Effects of heat stress on the renal and branchial carbohydrate metabolism and antioxidant system of Antarctic fish.

The objective of the present study was to assess the effect of short-term (2-144 h) heat stress (8 °C) on energy production processes and antioxidant defense systems in the kidneys and gills of Notothenia rossii and Notothenia coriiceps. Heat stress affected energy metabolism and oxidative stress parameters in a time-, tissue-, and species-dependent manner, and gills were more sensitive than kidneys to heat stress. N. rossii kidneys were able to stabilize carbohydrate metabolism after 12 h of heat stress, whereas the glycogen levels in N. coriiceps kidneys fluctuated in response to varying glucose-6-phosphatase (G6Pase) levels. The gills of N. rossii were able to stabilize their energy demand and aerobic metabolism under heat stress, whereas in the gills of N. coriiceps, changes in carbohydrate metabolic pathways depended on the exposure time: initially, anaerobiosis was activated after 6 h; the energy demand, characterized by glycogen consumption, increased after 72 h, and aerobic metabolism was activated within 144 h. With regard to the antioxidant defenses of the N. rossii kidney, it was found that levels of antioxidant enzymes were reduced during the first hours of heat stress, contributing to increased lipid peroxidation, whereas N. coriiceps kidneys did not show signs of oxidative damage. The gills of N. rossii exhibited more pronounced oxidative damage in response to heat stress than those of N. coriiceps despite the presence of increasing levels of antioxidants, likely due to tissue hypoxia.

Importance of the core structure of flavones in promoting inhibition of the mitochondrial respiratory chain.

Flavonoids are a large group of polyphenolic compounds that have received considerable attention because of their biological and physiological importance. The flavone (2-phenyl-4H-1-benzopyran-4one) used in this work is found in some cereal grains and generates several biological activities, including: apoptosis induction, cell cycle arrest, caspase activation and inhibition of tumor cell proliferation. However, its effects on the hepatic mitochondrial metabolism are still unknown. We evaluated the effect of flavone on the metabolism of mitochondria isolated from rat liver. Polarographic experiments using 200 micromol L(-1) flavone and rat liver mitochondria oxidizing glutamate or succinate indicated that both substrates underwent: (i) reduction of state 3 respiration; (ii) stimulation of state 4 respiration; (iii) reduction of the respiratory control coefficient; and (iv) reduction of the ADP/O ratio. An analysis of the activity of enzymatic complexes in the respiratory chain showed that flavone acts between complexes I and III. Flavone reduced the membrane electric potential at doses of 100, 150 and 200 micromol L(-1). Flavone at certain doses (75-200 micromol L(-1)) reduced mitochondrial swelling in the presence of valinomycin and KNO(3), suggesting that flavone could induce changes in mitochondrial membrane properties. These results demonstrate that the inhibition of mitochondrial enzymes in the respiratory chain coupled with the effects on membrane properties are promoted by the core structure of flavones, and these effects may be in part responsible for the cytotoxic effects of flavones.

Effects of natural flavones on membrane properties and citotoxicity of HeLa cells / Efeitos de flavonas naturais em propriedades de membranas e em citotoxicidade de células HeLa

The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM) decreased approximately 30 percent the rate and total amplitude of valinomycin induced swelling and 60-100 percent the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM) reduced 35 percent the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM) promoted a reduction of 40 percent in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.

O objetivo deste estudo foi avaliar se eupafolina e hispidulina, flavonas extraídas do Eupatorium littorale Cabrera, Asteraceae, possuíam a capacidade de alterar propriedades das membranas biológicas e promover efeitos citotóxicos. Eupafolina (50-200 µM) reduziu em aproximadamente 30 por cento a velocidade e amplitude do inchamento mitocondrial induzido por valinomicina e 60-100 por cento o inchamento mitocondrial dependente de substrato. Além disso, eupafolina na dose de 200 µM reduziu a transição de permeabilidade mitocondrial em 35 por cento entretanto, a hispidulina não alterou este parâmetro em todas as doses testadas. A avaliação da transição de fase dos lipossomas de DMPC com a sonda DPH demonstrou que ambas as flavonas afetam a fase gel e fluida. Quando lipossomas de membranas mitocondriais e a sonda DPH-PA foram utilizados, houve aumento da polarização de fluorescência promovido pela hispidulina. Eupafolina e hispidulina, na dose de 100 µM, promoveram 40 por cento de redução da viabilidade de células HeLa em 24 h. Nossos resultados sugerem que eupafolina e hispidulina têm efeitos citotóxicos que podem ser explicados em parte pelas alterações promovidas por estas flavonas sobre propriedades de membranas biológicas e sobre a bioenergética mitocondrial.

Eupafolin: Effect on mitochondrial energetic metabolism.

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.