REVIEW: Evidence supporting the 'preparation for oxidative stress' (POS) strategy in animals in their natural environment.

Hypometabolism is a common strategy employed by resilient species to withstand environmental stressors that would be life-threatening for other organisms. Under conditions such as hypoxia/anoxia, temperature and salinity stress, or seasonal changes (e.g. hibernation, estivation), stress-tolerant species down-regulate pathways to decrease energy expenditures until the return of less challenging conditions. However, it is with the return of these more favorable conditions and the reactivation of basal metabolic rates that a strong increase of reactive oxygen and nitrogen species (RONS) occurs, leading to oxidative stress. Over the last few decades, cases of species capable of enhancing antioxidant defenses during hypometabolic states have been reported across taxa and in response to a variety of stressors. Interpreted as an adaptive mechanism to counteract RONS formation during tissue hypometabolism and reactivation, this strategy was coined "Preparation for Oxidative Stress" (POS). Laboratory experiments have confirmed that over 100 species, spanning 9 animal phyla, apply this strategy to endure harsh environments. However, the challenge remains to confirm its occurrence in the natural environment and its wide applicability as a key survival element, through controlled experimentation in field and in natural conditions. Under such conditions, numerous confounding factors may complicate data interpretation, but this remains the only approach to provide an integrative look at the evolutionary aspects of ecophysiological adaptations. In this review, we provide an overview of representative cases where the POS strategy has been demonstrated among diverse species in natural environmental conditions, discussing the strengths and weaknesses of these results and conclusions.

Chaperone proteins: universal roles in surviving environmental stress.

Chaperone proteins have crucial roles to play in all animal species and are involved in mediating both the folding of newly synthesized peptides into their mature conformation, the refolding of misfolded proteins, and the trafficking of proteins between subcellular compartments. These highly conserved proteins have particularly important roles to play in dealing with disruptions of the proteome as a result of environmental stress since abiotic factors, including temperature, pressure, oxygen, water availability, and pollutants can readily disrupt the conformation and/or function of all types of proteins, e.g., enzymes, transporters, and structural proteins. The current review provides an update on recent advances in understanding the roles and responses of chaperones in aiding animals to deal with environmental stress, offering new information on chaperone action in supporting survival strategies including torpor, hibernation, anaerobiosis, estivation, and cold/freeze tolerance among both vertebrate and invertebrate species.

Hypoxia inducible factor-1α responds to freezing, anoxia and dehydration stresses in a freeze-tolerant frog.

The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, ∼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to ∼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state.

Regulation of the unfolded protein response during dehydration stress in African clawed frogs, Xenopus laevis.

The unfolded protein response (UPR) is a wide-ranging cellular response to accumulation of malfolded proteins in the endoplasmic reticulum (ER) and acts as a quality control mechanism to halt protein processing and repair/destroy malfolded proteins under stress conditions of many kinds. Among vertebrate species, amphibians experience the greatest challenges in maintaining water and osmotic balance, the high permeability of their skin making them very susceptible to dehydration and challenging their ability to maintain cellular homeostasis. The present study evaluates the involvement of the UPR in dealing with dehydration-mediated disruption of protein processing in the tissues of African clawed frogs, Xenopus laevis. This primarily aquatic frog must deal with seasonal drought conditions in its native southern Africa environment. Key markers of cellular stress that impact protein processing were identified in six tissues of frogs that had lost 28% of total body water, as compared with fully hydrated controls. This included upregulation of glucose-regulated proteins (GRPs) that are resident chaperones in the ER, particularly 2-ninefold increases in GRP58, GRP75, and/or GRP94 in the lung and skin. Activating transcription factors (ATF3, ATF4, ATF6) that mediate UPR responses also responded to dehydration stress, particularly in skeletal muscle where both ATF3 and ATF4 rose strongly in the nucleus. Other protein markers of the UPR including GADD34, GADD153, EDEM, and XBP-1 also showed selective upregulation in frog tissues in response to dehydration and nuclear levels of the transcription factors XBP-1 and P-CREB rose indicating up-regulation of genes under their control.

Mitochondria and the Frozen Frog.

The wood frog, Rana sylvatica, is the best-studied of a small group of amphibian species that survive whole body freezing during the winter months. These frogs endure the freezing of 65-70% of their total body water in extracellular ice masses. They have implemented multiple adaptations that manage ice formation, deal with freeze-induced ischemia/reperfusion stress, limit cell volume reduction with the production of small molecule cryoprotectants (glucose, urea) and adjust a wide variety of metabolic pathways for prolonged life in a frozen state. All organs, tissues, cells and intracellular organelles are affected by freeze/thaw and its consequences. This article explores mitochondria in the frozen frog with a focus on both the consequences of freezing (e.g., anoxia/ischemia, cell volume reduction) and mitigating defenses (e.g., antioxidants, chaperone proteins, upregulation of mitochondria-encoded genes, enzyme regulation, etc.) in order to identify adaptive strategies that defend and adapt mitochondria in animals that can be frozen for six months or more every year. A particular focus is placed on freeze-responsive genes in wood frogs that are encoded on the mitochondrial genome including ATP6/8, ND4 and 16S RNA. These were strongly up-regulated during whole body freezing (24 h at -2.5 °C) in the liver and brain but showed opposing responses to two component stresses: strong upregulation in response to anoxia but no response to dehydration stress. This indicates that freeze-responsive upregulation of mitochondria-encoded genes is triggered by declining oxygen and likely has an adaptive function in supporting cellular energetics under indeterminate lengths of whole body freezing.

Mitochondria, metabolic control and microRNA: Advances in understanding amphibian freeze tolerance.

Winter survival for many animal species depends freeze tolerance, a capacity to endure the conversion of as much as 65-70% of total body water into extracellular ice while reorganizing metabolism to provide cells with cryoprotection against insults that include prolonged ischemia and hyperosmotic stress. Natural freeze tolerance involves not just de novo preservation mechanisms such as synthesis of high levels of cryoprotectants or novel proteins that manage ice formation, but also requires attention to and co-ordination of many cellular processes. The present review examines recent studies of the freeze-tolerant wood frog (Rana sylvatica) that probed previously unexplored areas of metabolic adaptation for freezing survival, with a particular emphasis on mitochondria. Post-translational controls on enzyme function play a prominent role in resculpting metabolic responses of the wood frog to freezing including reversible phosphorylation control over fuel processing at the pyruvate dehydrogenase locus and modulation of antioxidant defense enzymes (Mn-SOD, catalase). Enzymes involved in mitochondrial nitrogen metabolism (glutamate dehydrogenase, carbamoyl phosphate synthetase) are also differentially regulated during freezing but by different post-translational modifications including ADP-ribosylation, lysine acetylation or glutarylation. The action of microRNAs in mediating post-translational controls on gene expression aid the suppression of energy-expensive (cell cycle) or destructive (apoptosis) processes in the frozen state while also providing storage of transcripts that will be immediately available for repair or reactivation of metabolic processes after thawing. The effects of low temperature in strengthening mRNA-microRNA interactions can also provide a passive mechanism of metabolic suppression in the frozen state.

In defense of proteins: Chaperones respond to freezing, anoxia, or dehydration stress in tissues of freeze tolerant wood frogs.

Wood frogs (Rana sylvatica LeConte) are the major model for studies of natural freeze tolerance by ectothermic vertebrates. Multiple biochemical adaptations support winter freezing survival but, to date, the protective role of chaperone proteins has received little attention. The present study analyzes responses to freezing, anoxia or dehydration exposures and recovery from these stresses by chaperone proteins in six wood frog organs: Five heat shock proteins (Hsc70, Hsp110, Hsp60, Hsp40, and Hsp10) and two glucose-regulated proteins (Grp78 and Grp94) were assessed. Hsc70 was upregulated in liver, muscle, heart and kidney (1.5-2.0 fold) during freezing and levels of its partner proteins also rose (Hsp110 in three tissues and Hsp40 in four tissues), these responses aligning most closely with comparable responses to anoxia rather than to dehydration. The resident chaperones of the endoplasmic reticulum (Grp78 and Grp94) also rose during freezing in liver and muscle (1.4-1.8 fold) but were suppressed in heart and skin, patterns that generally differed from responses to anoxia or dehydration. Elevated GRPs in liver may support the production and secretion of novel freeze responsive proteins. Increased levels of mitochondrial Hsp60 and Hsp10 (1.5-2.2 fold) occurred in most tissues during freezing and generally mimicked responses to anoxia. Overall, this study indicates that increased levels of chaperone proteins resident in multiple subcellular compartments contribute to stabilizing the cellular proteome during whole body freezing of wood frogs. These responses are probably derived from pre-existing amphibian defenses for stabilizing the proteome under environmental low oxygen or dehydration stresses.

Twenty years of the 'Preparation for Oxidative Stress' (POS) theory: Ecophysiological advantages and molecular strategies.

Freezing, dehydration, salinity variations, hypoxia or anoxia are some of the environmental constraints that many organisms must frequently endure. Organisms adapted to these stressors often reduce their metabolic rates to maximize their chances of survival. However, upon recovery of environmental conditions and basal metabolic rates, cells are affected by an oxidative burst that, if uncontrolled, leads to (oxidative) cell damage and eventually death. Thus, a number of adapted organisms are able to increase their antioxidant defenses during an environmental/functional hypoxic transgression; a strategy that was interpreted in the 1990s as a "preparation for oxidative stress" (POS). Since that time, POS mechanisms have been identified in at least 83 animal species representing different phyla including Cnidaria, Nematoda, Annelida, Tardigrada, Echinodermata, Arthropoda, Mollusca and Chordata. Coinciding with the 20th anniversary of the postulation of the POS hypothesis, we compiled this review where we analyze a selection of examples of species showing POS-mechanisms and review the most recent advances in understanding the underlying molecular mechanisms behind those strategies that allow animals to survive in harsh environments.

Interplay between diet-induced obesity and oxidative stress: Comparison between Drosophila and mammals.

Obesity caused by excessive fat accumulation in adipocytes is a growing global problem and is a major contributing risk factor for many chronic metabolic diseases. There is increasing evidence that oxidative stress plays a crucial role in both obesity progression and obesity-related complications. In recent years, Drosophila models of diet-induced obesity and associated pathologies have been successfully developed through manipulation of carbohydrate or fat concentrations in the food. Obese flies accumulate triacylglycerols in the fat body, an organ homologous to mammalian adipose tissue and exhibit metabolic and physiological complications including hyperglycemia, redox imbalance and shortened longevity; these are all similar to those observed in obese humans. In this review, we summarize current data on the mechanisms of oxidative stress induction in obesity, with emphasis on metabolic switches and the involvement of redox-responsive signaling pathways such as NF-κB and Nfr2. The recent achievements with D. melanogaster model suggest a complicated relationship between obesity, oxidative stress, and longevity but the Drosophila model offers probably the best opportunities to delve further into unraveling these interactions, particularly the roles of antioxidants and of Nrf2-regulated responses, in order to increase our understanding of the obese metabolic phenotype and test and develop anti-obesity pharmaceuticals.

Pesticide toxicity: a mechanistic approach.

Pesticides are known for their high persistence and pervasiveness in the environment, and along with products of their biotransformation, they may remain in and interact with the environment and living organisms in multiple ways, according to their nature and chemical structure, dose and targets. In this review, the classifications of pesticides based on their nature, use, physical state, pathophysiological effects, and sources are discussed. The effects of these xenobiotics on the environment, their biotransformation in terms of bioaccumulation are highlighted with special focus on the molecular mechanisms deciphered to date. Basing on targeted organisms, most pesticides are classified as herbicides, fungicides, and insecticides. Herbicides are known as growth regulators, seedling growth inhibitors, photosynthesis inhibitors, inhibitors of amino acid and lipid biosynthesis, cell membrane disrupters, and pigment biosynthesis inhibitors, whereas fungicides include inhibitors of ergosterol biosynthesis, protein biosynthesis, and mitochondrial respiration. Insecticides mainly affect nerves and muscle, growth and development, and energy production. Studying the impact of pesticides and other related chemicals is of great interest to animal and human health risk assessment processes since potentially everyone can be exposed to these compounds which may cause many diseases, including metabolic syndrome, malnutrition, atherosclerosis, inflammation, pathogen invasion, nerve injury, and susceptibility to infectious diseases. Future studies should be directed to investigate influence of long term effects of low pesticide doses and to minimize or eliminate influence of pesticides on non-target living organisms, produce more specific pesticides and using modern technologies to decrease contamination of food and other goods by pesticides.

Acute exposure to copper induces variable intensity of oxidative stress in goldfish tissues.

Copper is an essential element, but at high concentrations, it is toxic for living organisms. The present study investigated the responses of goldfish, Carassius auratus, to 96 h exposure to 30, 300, or 700 µg L-1 of copper II chloride (Cu2+). The content of protein carbonyls was higher in kidney (by 158%) after exposure to 700 mg L-1 copper, whereas in gills, liver, and brain, we observed lower content of protein carbonyls after exposure to copper compared with control values. Exposure to copper resulted in increased levels of lipid peroxides in gills (76%) and liver (95-110%) after exposure to 300 and 700 µg L-1 Cu2+. Low molecular mass thiols were depleted by 23-40% in liver and by 29-67% in kidney in response to copper treatment and can be used as biomarkers toxicity of copper. The activities of primary antioxidant enzymes, superoxide dismutase and catalase, were increased in liver as a result of Cu2+ exposure, whereas in kidney catalase activity was decreased. The activities of glutathione-related enzymes, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase were decreased as a result of copper exposure, but glutathione reductase activity increased by 25-40% in liver. Taken together, these data show that exposure of fish to Cu2+ ions results in the development of low/high intensity oxidative stress reflected in enhanced activities of antioxidant and associated enzymes in different goldfish tissues.

Dietary L-arginine accelerates pupation and promotes high protein levels but induces oxidative stress and reduces fecundity and life span in Drosophila melanogaster.

L-Arginine, a precursor of many amino acids and of nitric oxide, plays multiple important roles in nutrient metabolism and regulation of physiological functions. In this study, the effects of L-arginine-enriched diets on selected physiological responses and metabolic processes were assessed in Drosophila melanogaster. Dietary L-arginine at concentrations 5-20 mM accelerated larval development and increased body mass, and total protein concentrations in third instar larvae, but did not affect these parameters when diets contained 100 mM arginine. Young (2 days old) adult flies of both sexes reared on food supplemented with 20 and 100 mM L-arginine possessed higher total protein concentrations and lower glucose and triacylglycerol concentrations than controls. Additionally, flies fed 20 mM L-arginine had higher proline and uric acid concentrations. L-Arginine concentration in the diet also affected oxidative stress intensity in adult flies. Food with 20 mM L-arginine promoted lower protein thiol concentrations and higher catalase activity in flies of both sexes and higher concentrations of low molecular mass thiols in males. When flies were fed on a diet with 100 mM L-arginine, lower catalase activities and concentrations of protein thiols were found in both sexes as well as lower low molecular mass thiols in females. L-Arginine-fed males demonstrated higher climbing activity, whereas females showed higher cold tolerance and lower fecundity, compared with controls. Food containing 20 mM L-arginine shortened life span in both males and females. The results suggest that dietary L-arginine shows certain beneficial effects at the larval stage and in young adults. However, the long-term consumption of L-arginine-enriched food had unfavorable effects on D. melanogaster due to decreasing fecundity and life span.

High amylose starch consumption induces obesity in Drosophila melanogaster and metformin partially prevents accumulation of storage lipids and shortens lifespan of the insects.

There are very few studies that have directly analyzed the effects of dietary intake of slowly digestible starches on metabolic parameters of animals. The present study examined the effects of slowly digestible starch with high amylose content (referred also as amylose starch) either alone, or in combination with metformin on the development, lifespan, and levels of glucose and storage lipids in the fruit fly Drosophila melanogaster. Consumption of amylose starch in concentrations 0.25-10% did not affect D. melanogaster development, whereas 20% starch delayed pupation and reduced the number of larvae that reached the pupal stage. Starch levels in larval food, but not in adult food, determined levels of triacylglycerides in eight-day-old adult flies. Rearing on diet with 20% starch led to shorter lifespan and a higher content of triacylglycerides in the bodies of adult flies as compared with the same parameters in flies fed on 4% starch diet. Food supplementation with 10mM metformin partly attenuated the negative effects of high starch concentrations on larval pupation and decreased triacylglyceride levels in adult flies fed on 20% starch. Long-term consumption of diets supplemented with metformin and starch decreased lifespan of the insects, compared with the diet supplemented with starch only. The data show that in Drosophila high starch consumption may induce a fat fly phenotype and metformin may partially prevent it.

Elevated chaperone proteins are a feature of winter freeze avoidance by larvae of the goldenrod gall moth, Epiblema scudderiana.

Winter survival for many insect species includes a need to maintain metabolic homeostasis and structural/functional integrity of macromolecules not only over a wide range of cold temperatures but also in response to rapid temperature change. Chaperones are well-known to protect/stabilize protein structure with regard to heat stress but less is known about their potential involvement in long-term protection of the proteome at subzero temperatures. The present study assessed the participation of chaperone proteins in the cold hardiness of larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae), monitoring changes in nine proteins over the winter months as well as their responses to laboratory cold acclimation or anoxia exposure. Four heat shock proteins (HSPs: Hsp110, Hsp70, Hsp60, Hsp40), three glucose-regulated proteins (GRPs: Grp78, Grp 94, Grp170) and the tailless complex polypeptide 1 (TCP-1) as well as the heat shock transcription factor (HSF1) were investigated. In general, all were significantly elevated in larvae collected from an outdoor site between October and March, as compared with September values, and chaperone levels were reduced again in April. The October to March interval also includes the period of diapause followed by cold quiescence in the species. Relative expression of Hsp70, Hsp60 and Hsp40 rose by 2-3-fold, GRPs increased 1.5-3-fold, and levels of active (hyperphosphorylated) HSF1 increased by 4-4.8-fold over the midwinter months. Chilling from 15°C to 4°C in the laboratory upregulated Grp78 protein content that remained high as temperature was further reduced to -4°C and then -20°C whereas Hsp110, Hsp70 and HSF1 levels increased when larvae were exposed to -4°C and -20°C. Grp170 (also known as oxygen-regulated protein 150) was the only chaperone that increased significantly in the larvae in response to anoxia exposure at 4°C. The data also indicated that multiple subcellular compartments received enhanced protection for their proteome since upregulation of chaperones included proteins known to occur in cytosolic (Hsp40, Hsp70), mitochondrial (Hsp60) and endoplasmic reticulum (Grp170) locations. Overall, the data indicate that chaperones have a significant role to play in the winter cold hardiness of E. scudderiana and identify declining temperatures (and perhaps also oxygen restriction) as potential modulators of chaperone production. The data add support to a relatively understudied area of insect cold hardiness - the long-term protection and stabilization of the proteome over the winter months.

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster.

OBJECTIVES: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na2MoO4). METHODS: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food. RESULTS: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration. DISCUSSION: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.

Molecular Physiology of Freeze Tolerance in Vertebrates.

Freeze tolerance is an amazing winter survival strategy used by various amphibians and reptiles living in seasonally cold environments. These animals may spend weeks or months with up to ∼65% of their total body water frozen as extracellular ice and no physiological vital signs, and yet after thawing they return to normal life within a few hours. Two main principles of animal freeze tolerance have received much attention: the production of high concentrations of organic osmolytes (glucose, glycerol, urea among amphibians) that protect the intracellular environment, and the control of ice within the body (the first putative ice-binding protein in a frog was recently identified), but many other strategies of biochemical adaptation also contribute to freezing survival. Discussed herein are recent advances in our understanding of amphibian and reptile freeze tolerance with a focus on cell preservation strategies (chaperones, antioxidants, damage defense mechanisms), membrane transporters for water and cryoprotectants, energy metabolism, gene/protein adaptations, and the regulatory control of freeze-responsive hypometabolism at multiple levels (epigenetic regulation of DNA, microRNA action, cell signaling and transcription factor regulation, cell cycle control, and anti-apoptosis). All are providing a much more complete picture of life in the frozen state.

Acute exposure to the penconazole-containing fungicide Topas partially augments antioxidant potential in goldfish tissues.

Penconazole is a systemic fungicide commonly used in agriculture as the commercial preparation Topas. Although triazole fungicides are widely found in the aquatic environment, little is known about their acute toxicity on fish. In this study we assessed the effects of short-term exposure to Topas on some parameters of homeostasis of reactive oxygen species (ROS), such as the levels of markers of oxidative stress and parameters of the antioxidant defense system of goldfish (Carassius auratus L.). Gills appeared to be the main target organ of Topas toxicity, showing the greatest number of parameters affected. Gills of Topas-treated fish showed a higher content of low (L-SH) and high (H-SH) molecular mass thiols and higher activities of superoxide dismutase (SOD), catalase, glutathione reductase (GR), glutathione-S-transferase (GST), and glucose-6-phosphate dehydrogenase (G6PDH) as well as reduced carbonyl protein content (CP), as compared with those in the control group. In the liver, goldfish exposure to 15-25mgL-1 Topas resulted in a higher L-SH and H-SH content, but lower CP levels and activity of GST. In kidney, Topas exposure resulted in higher activities of glutathione peroxidase (GPx) and G6PDH, but lower L-SH content and activity of GST. The results of this study indicate that acute goldfish exposure to the triazole fungicide Topas increased efficiency of the antioxidant system in fish gills, liver, and kidney. This could indicate the development of low intensity oxidative stress which up-regulates defense mechanisms responsible for protection of goldfish against deleterious ROS effects.

Dietary alpha-ketoglutarate promotes higher protein and lower triacylglyceride levels and induces oxidative stress in larvae and young adults but not in middle-aged Drosophila melanogaster.

Alpha-ketoglutarate (AKG) is involved in multiple metabolic and regulatory pathways. In this work, the effects of AKG-supplemented diets on selected physiological responses and metabolic processes, including metabolism of reactive oxygen species, was assessed in larvae and adult (both 2 and 24days old) Drosophila melanogaster. Dietary supplementation with AKG resulted in dose-dependent effects on larval development, body composition and antioxidant status of third instar larvae. Larvae and young (2days post-eclosion) adult females fed on AKG shared similar metabolic changes such as higher total protein levels, lower triacylglyceride levels and higher values for oxidative stress indices, namely lipid peroxides and low molecular mass thiols. The latter indicated the development of oxidative stress which, in turn, may induce adaptive responses that can explain the higher resistance of AKG-fed young females to heat shock and hydrogen peroxide exposure. In contrast to young flies, middle-aged females (24days) on AKG-containing diet possessed higher total protein, glucose and triacylglyceride levels, whereas oxidative stress parameters were virtually the same as compared with control females of the same age. In parallel, females fed an AKG-supplemented diet showed lower fecundity, higher heat shock resistance but no change in oxidative stress resistance at middle age which in combination with levels of protein, glucose, and triacylglycerides can be considered as potentially beneficial AKG effects for aging organisms. To our best knowledge, this is the first study on age-matched AKG influence on animals' organism which shows that Drosophila may be used as a model for previous quick study in cost-efficient manner age-related AKG effects in mammals and humans.

Oxidative stress responses in gills of goldfish, Carassius auratus, exposed to the metribuzin-containing herbicide Sencor.

Metribuzin belongs to the family of asymmetrical triazine compounds and is an active ingredient in many commercial herbicides including Sencor. Effects on goldfish (Carassius auratus L.) of exposure for 96h to 7.14, 35.7 or 71.4mgL(-1) Sencor 70 WG (corresponding to 5, 25 and 50mgL(-1) of metribuzin) were examined by evaluating oxidative stress markers and activities of antioxidant and associated enzymes in gills. Fish exposed to the lowest Sencor concentration (7.14mgL(-1)) showed a 94% increase in levels of protein carbonyls in gills as well as 45% and 144% increases in the activities of glutathione peroxidase and glutathione-S-transferase. Exposure to the highest Sencor concentration (71.4mgL(-1)) resulted in reduced levels of protein carbonyls by 56% and lipid peroxides by 40%, as compared with controls, but enhanced levels of low and high molecular mass thiols by 71% and 36%, respectively. The activities of superoxide dismutase, glutathione peroxidase and glutathione-S-transferase were increased in gills of goldfish exposed to 71.4mgL(-1) Sencor. At any concentration tested, Sencor did not affect the activities of glutathione reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase or acetylcholine esterase in gills. The results of this study indicate that acute exposure of goldfish to Sencor had effect on free radical processes in gills and glutathione-dependent antioxidants effectively protect proteins and lipids from oxidation.

Alpha-ketoglutarate attenuates toxic effects of sodium nitroprusside and hydrogen peroxide in Drosophila melanogaster.

The protective effects of dietary alpha-ketoglutarate (AKG) are described that aid fruit flies, Drosophila melanogaster, to resist sodium nitroprusside (SNP) and hydrogen peroxide toxicity. Food supplementation with 10mM AKG alleviated toxic effects of 1mM SNP added to food and improved fly development. Dietary AKG also prevented the increase in levels of oxidative stress markers seen in SNP-reared adult flies. In vitro AKG did not affect the rate of SNP decomposition and did not bind iron and nitrite ions released in this process. Alpha-ketoglutarate also displayed high H2O2-scavenging activity in vitro and efficiently protected adult flies against this compound in combined treatments. Based on the observed antioxidant activity of AKG, it may be suggested that the antioxidant mode of AKG action (apart from its cyanide-binding capability) may be used to prevent the toxic effects of SNP and improve general physiological state of D. melanogaster and other animals and humans.