Daniel C. Moreira

Glutathione status and antioxidant enzymes in a crocodilian species from the swamps of the Brazilian Pantanal

In a previous study oxidative damage markers - lipid peroxidation and protein oxidation - were determined in organs of wild Caiman yacare captured in winter-2001 and summer-2002 at various developmental stages. An increase in oxidative damage occurred in the hatchling-juvenile transition (but not in the juvenile-adult transition) and winter-summer transition (in juveniles), suggesting that oxidative stress is associated with development and season. Herein the effect of development and season on glutathione (GSH) metabolism and the effect of development on the activity of antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase) and glucose 6-phosphate dehydrogenase were analyzed. The ratio GSSG:GSH-eq increased in lung, liver, kidney and brain by 1.8- to 4-fold in the embryo/hatchling to juvenile transition. No changes occurred in juvenile-adult transition. GSSG:GSH-eq across seasons was significantly elevated in summer. Total-glutathione content was mostly stable in various organs; in liver it increased in the embryo-juvenile transition. Enzyme activities were only determined in summer-animals (embryos, hatchlings and juveniles). For most antioxidant enzymes, activities increased from embryo/hatchling to juvenile in liver and Kidney. In lung, there was an inverse trend for enzyme activities and total glutathione content. Thus, increased metabolic rates during early caiman growth - in embryo-juvenile transition - appears to be related to redox imbalance as suggested by increased GSSG:GSH-eq and activation of antioxidant defenses. Differences in oxidative stress across seasons were related with summer-winter nocturnal temperatures. These results, as a whole, were interpreted in the context of ecological biochemistry.

How widespread is preparation for oxidative stress in the animal kingdom

Abstract It is well known that many anoxia/hypoxia tolerant species when exposed to anoxia/hypoxia respond by increasing the activity/expression of antioxidant enzymes and/or glutathione levels—a phenomenon called “preparation for oxidative stress” (POS). This phenomenon was also observed during freezing exposure, severe dehydration, aerial exposure of water-breathing animals and estivation. However, as far as we know, there is no analysis available of the prevalence of POS among animal species. A major problem is the very definition of POS, since many animal species show both increases and decreases of antioxidants during low oxygen stress and estivation. Therefore, we established three different criteria; from inclusive to restrictive and analyzed how widespread the POS phenomenon is in the animal kingdom. We analyzed all available papers in several databases about the modulation of antioxidant defenses during oxygen deprivation or estivation. Based on the magnitude of change (as % change) during the specific low oxygen stresses or estivation, we classified each species as POS-positive, POS-negative or POS-neutral, considering the three different criteria. The prevalence of POS-positive animals (102 species from 8 phyla) was stress-dependent: in estivation and dehydration it was 91–100%, while in hypoxia it was 37.5–53%, depending on the criteria. In the case of air exposure, anoxia and freezing the proportions of POS-positive species were 54–77%, 64–77% and 75–86%, respectively. Overall, the prevalence of POS was 58 to 68% when all stresses and all species were analyzed together. The results indicate the key importance of POS as a survival strategy of animals exposed to freezing, dehydration and estivation, and, to a lesser extent, to oxygen deprivation itself (i.e. hypoxia and anoxia).

Ellagic acid inhibits iron-mediated free radical formation.

Polyphenols are reported to have some health benefits, which are link to their antioxidant properties. In the case of ellagic acid (EA), there is evidence that it has free radical scavenger properties and that it is able to form complexes with metal ions. However, information on a possible link between the formation of iron-EA complexes and their interference in Haber-Weiss/Fenton reactions was not yet determined. Thus, the present study investigated the in vitro antioxidant mechanism of EA in a system containing ascorbate, Fe(III) and different iron ligands (EDTA, citrate and NTA). Iron-mediated oxidative degradation of 2-deoxyribose was poorly inhibited (by 12%) in the presence of EA (50μM) and EDTA. When citrate or NTA - which form weak iron complexes - were used, the 2-deoxyribose protection increased to 89-97% and 45%, respectively. EA also presented equivalent inhibitory effects on iron-mediated oxygen uptake and ascorbyl radical formation. Spectral analyses of iron-EA complexes show that EA removes Fe(III) from EDTA within hours, and from citrate within 1min. This difference in the rate of iron-EA complex formation may explain the antioxidant effects of EA. Furthermore, the EA antioxidant effectiveness was inversely proportional to the Fe(III) concentration, suggesting a competition with EDTA. In conclusion, the results indicate that EA may prevent in vitro free radical formation when it forms a complex with iron ions.

Current Trends and Research Challenges Regarding “Preparation for Oxidative Stress”

Survival under stress, such as exposure to hypoxia, anoxia, freezing, dehydration, air exposure of water breathing organisms, and estivation, is commonly associated to enhanced endogenous antioxidants, a phenomenon coined “preparation for oxidative stress” (POS). The regulation of free radical metabolism seems to be crucial under these selective pressures, since this response is widespread among animals. A hypothesis of how POS works at the molecular level was recently proposed and relies on two main processes: increased reactive species production under hypoxia, and activation of redox-sensitive transcription factors and signaling pathways, increasing the expression of antioxidants. The present paper brings together the current knowledge on POS and considers its future directions. Data indicate the presence of POS in 83 animal species (71.6% among investigated species), distributed in eight animal phyla. Three main research challenges on POS are presented: (i) to identify the molecular mechanism(s) that mediate/induce POS, (ii) to identify the evolutionary origins of POS in animals, and (iii) to determine the presence of POS in natural environments. We firstly discuss the need of evidence for increased RS production in hypoxic conditions that underlie the POS response. Secondly, we discuss the phylogenetic origins of POS back 700 million years, by identifying POS-positive responses in cnidarians. Finally, we present the first reports of the POS adaptation strategy in the wild. The investigation of these research trends and challenges may prove useful to understand the evolution of animal redox adaptations and how they adapt to increasing stressful environments on Earth.

Structure and function of a novel antioxidant peptide from the skin of tropical frogs

ABSTRACT The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin‐I, has a molecular weight [M+H]+ = 1543.69 Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin‐I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin‐I has a novel primary sequence with low similarity compared with previously described amphibians AOPs. Antioxidin‐I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin‐I presented a low cytotoxicity and suppressed menadione‐induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia‐induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide. Graphical abstract Figure. No caption available. HighlightsAntioxidin‐I is a new antioxidant peptide isolated from the skin tropical frogs.The bioactive peptide presented very low cytotoxicity against mammalian cells.It was able to avoid redox imbalance in oxidative challenged cells.Antioxidin‐I had the capacity to suppress ROS levels in hypoxia‐exposed microglia.Results support the application of the peptide for neuroprotection.

Redox Metabolism During Tropical Diapause in a Lepidoptera Larva

Many studies on metabolic rate depression and redox metabolism exist in the literature; however, virtually none focuses on tropical insect diapause. Thus, our aim was to evaluate peculiarities of the metabolism of reactive oxygen species (ROS) between diapausing and non-diapausing insects in a tropical region. The lepidopteran Chlosyne lacinia undergoes diapause as larva at the third instar prior to the dry season in middle-west Brazil. We measured the activity of metabolic and anti-oxidant enzymes at day 20 of diapause. The activity of citrate synthase decreased by 81% in whole-body extracts as compared with larvae sampled before diapause entry. Moreover, total-glutathione content and lipid peroxidation dropped significantly (by 82 and 24%, respectively) in diapausing insects. On the other hand, the activities of catalase and glucose 6-phosphate dehydrogenase (G6PDH) were unchanged. These results indicate a diminished oxidative metabolism and suggest important roles for catalase and G6PDH in ROS control in diapause and, possibly, during arousal. The diminished glutathione levels could be related to its depletion by glutathione-dependent systems or by its diminished biosynthesis.

Antioxidant activity and mechanism of commercial Rama Forte persimmon fruits (Diospyros kaki)

This study aimed to characterize the antioxidant properties of Rama Forte persimmon, a tannin-rich fruit variety produced in Brazil. Extracts prepared with lyophilized pulps from fruits obtained in local markets were analyzed individually to evaluate the extent of antioxidant protection and investigate the antioxidant mechanism. Iron-mediated hydroxylation of 5,5-dimethyl-1-pirrolidine-N-oxide, determined by electron paramagnetic resonance (EPR), and oxidative degradation of 2-deoxyribose (2-DR) were inhibited by fruit extracts in a dose-dependent manner. There was a considerable individual variability in inhibition of 2-DR degradation by individual fruits. Higher protection of 2-DR degradation (by the extracts) was observed in Fe(III)-citrate/ascorbate in comparison with Fe(III)-EDTA/ascorbate system; however, antioxidant effectiveness of fruit extracts was not diminished by increasing EDTA concentration by 10-fold. Other competition experiments using the 2-DR assay (varying pre-incubation time and 2-DR concentration) indicated that protection comes mainly from free radical scavenging, rather that metal chelation antioxidant activity. Persimmon extracts prevented iron-mediated lipid peroxidation in rat liver homogenates, which correlated significantly with the inhibition of 2-DR oxidation. Finally, sugar content of individual fruits correlated inversely with inhibition of 2-DR degradation, which could indicate that maturation decreases soluble antioxidant concentration or efficiency. In conclusion, lipid peroxidation, 2-DR and EPR experiments indicated that extracts from commercial fruits showed mainly radical-scavenger activity and relevant antioxidant activity.

Structure–Activity Relationship of Piplartine and Synthetic Analogues against Schistosoma mansoni and Cytotoxicity to Mammalian Cells

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure–function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5–10 µM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.

Corrigendum: Current Trends and Research Challenges Regarding “Preparation for Oxidative Stress”

[This corrects the article DOI: 10.3389/fphys.2017.00702.].

Is “Preparation for Oxidative Stress” a Case of Physiological Conditioning Hormesis?

Many animal species endure hypoxic or even anoxic stresses, when faced with harsh environmental conditions including freezing, severe dehydration and air exposure of aquatic organisms. Hypoxia in those animals induces a set of physiological/biochemical adaptive responses, allowing organisms to cope with low oxygen levels. Such responses are mediated by (i) arrest of transcriptional and translational activity, (ii) depression of metabolic rate, (iii) re-wiring of energy metabolism pathways toward fermentative rather than oxidative routes, (iv) activation of mechanisms involved in both macromolecular repair and detoxification of cellular-derived oxidants (Storey and Storey, 2011; Storey, 2015). In this regard, a transient up-regulation of endogenous antioxidant enzymes aiming the improvement of reactive species (RS) detoxification has emerged as a hallmark for many organisms to tolerate hypoxic stresses. Such phenomenon was coined “preparation for oxidative stress” (POS) 20 years ago, and numerous examples have supported POS as a physiological mechanism to deal with environmental stresses (Hermes-Lima and Storey, 1995, 1996; Hermes-Lima et al., 1998, 2015; Hermes-Lima and Zenteno-Savin, 2002; Lushchak et al., 2005; Welker et al., 2013). So far, we have identified POS as an adaptive physiological mechanism in 83 animal species from 8 different phyla when exposed to low oxygen stress and during estivation (Moreira et al., 2016, 2017). The phenotypes generated by POS include the up-regulation of superoxide dismutase (SOD), catalase and glutathione transferase (GST) activities by ~80% in Otala lactea snails during estivation (Hermes-Lima and Storey, 1995). Interestingly, snails that return to active state decrease all antioxidant enzyme activities to pre-estivation levels. Similar observations were reported when Rana pipiens frogs were challenged with 30 h anoxia, causing transient catalase, and GST activation (Hermes-Lima and Storey, 1996). Also, transient increases of catalase and glutathione peroxidase (GPX) activities by 30–70% were observed in the brain of common carp during hypoxia (Lushchak et al., 2005). Increases by ~60% in muscular SOD activity were also observed in Lacerta vivipara lizards upon freezing, which returns to control levels after thawing (Voituron et al., 2006). Evidence suggests the existence of common mechanisms underlying dormancy states induced by hypoxia, hypoxic-like conditions and aerobic hypometabolism. For example, it is known that hypoxia maintains the redox state of mitochondrial electron transport system (ETS) toward a reduced state, favoring the production of superoxide radicals (Chandel et al., 1998; Vanden Hoek et al., 1998; Hernansanz-Agustin et al., 2014). Thus, against the common-sense, reduced oxygenation increases, rather than decreases, cellular oxidants production (Murphy, 2009; Smith et al., 2017; see legend of Figure ​Figure1A).1A). Accordingly, the proposed mechanism by which POS confers tolerance to oxidant insults, considers an increase in mitochondrial RS formation during low oxygen stress, followed by redox imbalance that activates redox-sensitive transcription factors, such as NF-κB, FoxOs, and Nrf2 (Schreck et al., 1991; Ishii et al., 2000; Essers et al., 2004). Additionally, redox imbalance also shifts protein phosphorylation levels toward a higher phosphorylated state, by either reducing protein phosphatase and/or increasing protein kinase activities (Staal et al., 1994; Meng et al., 2002; Howe et al., 2004; Corcoran and Cotter, 2013) (Figure ​(Figure1A).1A). In this regard, oxidants can inhibit multiple protein tyrosine phosphatases including PTP1B and PTEN (Leslie et al., 2003; Salmeen et al., 2003), with direct consequences to cell function. Conversely, oxidant conditions activate several protein kinases such as Src (Devary et al., 1992), MAPK (Goldstone and Hunt, 1997) and calcium/calmodulin-dependent protein kinases (Howe et al., 2004). However, it seems that maintenance of the higher phosphorylated state of protein targets by redox imbalance may occur through protein phosphatase inhibition rather than direct protein kinase activation by oxidants (Lee and Esselman, 2002). The consequences of higher protein phosphorylation to cellular redox homeostasis are: (i) the activation of redox-sensitive transcription factors (Shirakawa and Mizel, 1989), and/or (ii) regulation of antioxidant enzymes activities by direct phosphorylation. Examples include the demonstration that Nrf2 expression depends on low PTEN phosphatase activity, rendering tumor cells more proliferative (Rojo et al., 2014). Likewise, maintenance of oxidant conditions indirectly activates antioxidant enzymes through their phosphorylation, acting independently of redox-sensitive transcription factors (Rhee and Woo, 2011; Rafikov et al., 2014; Tsang et al., 2014). Ultimately, higher tolerance to multiple redox stresses is afforded by increasing endogenous antioxidant levels mediated by either activation of redox-sensitive transcription factors or by activation of antioxidant enzymes through phosphorylation or other covalent modifications (Figure ​(Figure1A1A). Open in a separate window Figure 1 Molecular oxygen is absolutely required for maintenance of cellular energy and redox homeostasis across different animal species. Although some organisms cannot tolerate slight hypoxia, others can adapt to and survive strong shortages in oxygen supply even for long periods of time. A common trend observed in some hypoxia-tolerant animals is their enhanced capacity to boost antioxidant defenses during a number of stresses, a phenomenon known as “preparation for oxidative stress” (POS). POS was identified in animals from 8 distinct phyla and despite the molecular mechanisms are not fully understood, we have recently proposed an explanation (Hermes-Lima et al., 2015), where the role of phosphatases and kinases in POS is highlighted herein, as well as the increased cellular oxidant production under hypoxia (A). During hypoxia, the redox state of ETS and mitochondrial dehydrogenases shifts toward a reduced state due to limited electron transfer from cytochrome c oxidase to oxygen. This leads to increased electron availability in many enzymes/complexes involved in redox reactions, consequently favoring superoxide production (Smith et al., 2017). Importantly, given that a very small percentage of molecular oxygen is converted to superoxide in isolated mammalian mitochondria (about 0.2%, Tahara et al., 2009) and that this figure is likely to be much lower in vivo (Murphy, 2009), it is suggestive that the electron availability, not oxygen concentration, would be the limiting factor in mitochondrial superoxide production (Campian et al., 2007). Therefore, even in hypoxia, increases in electron availability should boost mitochondrial superoxide production—at least until molecular oxygen concentration becomes so low that electron availability ceases to be the limiting factor. Thus, the overall pattern observed is an increase in oxidant formation during hypoxia. The pattern of transient activation of antioxidant defenses along hypoxic challenges, and the improved protection against stressful insults generated afterwards, follows the same trend observed in many cases of physiological conditioned hormesis. Limited time and magnitude exposure of animals to insults including hypoxia/anoxia, freezing and severe dehydration, as well as to conditions inducing estivation, activates a “physiological program” that reduces adaptive failure and/or mortality upon stronger challenges (the “hormetic zone”), as proposed in the hormesis concept. “Conditioned re-oxygenation” (or reoxygenation-like, during dehydration/rehydration and freezing/thawing), shown in (B), is a state where the protective POS-response range is maximum. However, longer and/or stronger exposure to these insults revert the protective hormetic effects (the “harmful zone”), increasing adaptive failure. Therefore, given their remarkable similarities in biological and biochemical outputs, we propose that POS should be included as a new example of physiological conditioning hormesis. Graphic elements adapted from Servier Medical Art.