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.

Commentary: On the merit of an early contributor of the "Preparation for Oxidative Stress" (POS) theory.

This commentary acknowledges the contributions of the Ukrainian biologist, Dr. Volodymyr Lushchak, to the understanding of the physiological adaptive strategy called "Preparation for Oxidative Stress" (POS). In the 1990s, various studies revealed that activities of antioxidant enzymes rose in animals under hypometabolic conditions. These timely observations allowed scientists to propose that this increase could prepare animals for reoxygenation events following the release of oxygen restriction, but in doing so, would trigger oxidative damage, hence the use of the term "preparation". Over next 25 years, the phenomenon was described in detail in more than one hundred studies of animals under conditions of aestivation, hypoxia/anoxia, freezing, severe dehydration, ultraviolet exposure, air exposure of water-breathing animals, salinity stress, and others. The POS phenomenon remained without a mechanistic explanation until 2013, when it was proposed that a small increase in oxyradical formation during hypoxia exposure (in hypoxia-tolerant animals) could activate redox-sensitive transcription factors that, in turn, would initiate transcription and translation of antioxidant enzymes. Dr. Lushchak, who studied goldfish under severe hypoxia in the 1990s, had actually proposed the increased production of oxyradicals under this condition and concluded that it would lead to an upregulation of antioxidant enzymes, the hallmark of the POS strategy. However, his research partner at the time, Dr. Hermes-Lima, thought the idea did not have sufficient evidence to support it and recommended the removal of this explanation. In those days, the main line of thinking was that increased oxyradical formation under hypoxia was "impossible". So, as it turns out, the ideas of Dr. Lushchak were well ahead of his time. It then took >10 years before the biochemical and molecular mechanisms responsible for triggering the POS response were clarified. In the present article, this fascinating history is described to highlight Dr. Lushchak's contributions and insights about the POS theory.

Corrigendum: Current Trends and Research Challenges Regarding "Preparation for Oxidative Stress".

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

Subtropical hibernation in juvenile tegu lizards (Salvator merianae): insights from intestine redox dynamics.

Juvenile tegu lizards (Salvator merianae) experience gradual and mild temperature changes from autumn to winter in their habitat. This tropical/subtropical reptile enter a state of dormancy, with an 80% reduction in metabolic rate, that remains almost constant during winter. The redox metabolism in non-mammalian vertebrates that hibernate under such distinguished conditions is poorly understood. We analyzed the redox metabolism in the intestine of juvenile tegus during different stages of their first annual cycle. The effect of food deprivation (in spring) was also studied to compare with fasting during hibernation. Both winter dormancy and food deprivation caused decreases in reduced glutathione levels and glutathione transferase activity. While glutathione peroxidase and glutathione transferase activities decreased during winter dormancy, as well as glutathione (GSH) levels, other antioxidant enzymes (catalase, superoxide dismutase and glutathione reductase) remained unchanged. Notably, levels of disulfide glutathione (GSSG) were 2.1-fold higher in late autumn, when animals were in the process of depressing metabolism towards hibernation. This increased "oxidative tonus" could be due to a disruption in NADPH-dependent antioxidant systems. In dormancy, GSSG and lipid hydroperoxides were diminished by 60-70%. The results suggest that the entrance into hibernation is the main challenge for the redox homeostasis in the intestine of juvenile tegus.

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.

Blog construction as an effective tool in biochemistry active learning.

To boost active learning in undergraduate students, they were given the task of preparing blogs on topics of clinical biochemistry. This "experiment" lasted for 12 teaching-semesters (from 2008 to 2013), and included a survey on the blogs' usefulness at the end of each semester. The survey (applied in the 2008-2010 period) used a Likert-like questionnaire with eight questions and a 1-to-6 scale, from "totally disagree" to "fully agree." Answers of 428 students were analyzed and indicated overall approval of the blog activity: 86% and 35% of the responses scored 4-to-6 and 6, respectively. Considering the survey results, the high grades obtained by students on their blogs (averaging 8.3 in 2008-2010), and the significant increase in average grades of the clinical biochemistry exam after the beginning of the blog system (from 5.5 in 2007 to 6.4 in 2008-2010), we concluded that blogging activity on biochemistry is a promising tool for boosting active learning. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):205-215, 2017.

Preparation for oxidative stress under hypoxia and metabolic depression: Revisiting the proposal two decades later.

Organisms that tolerate wide variations in oxygen availability, especially to hypoxia, usually face harsh environmental conditions during their lives. Such conditions include, for example, lack of food and/or water, low or high temperatures, and reduced oxygen availability. In contrast to an expected strong suppression of protein synthesis, a great number of these animals present increased levels of antioxidant defenses during oxygen deprivation. These observations have puzzled researchers for more than 20 years. Initially, two predominant ideas seemed to be irreconcilable: on one hand, hypoxia would decrease reactive oxygen species (ROS) production, while on the other the induction of antioxidant enzymes would require the overproduction of ROS. This induction of antioxidant enzymes during hypoxia was viewed as a way to prepare animals for oxidative damage that may happen ultimately during reoxygenation. The term "preparation for oxidative stress" (POS) was coined in 1998 based on such premise. However, there are many cases of increased oxidative damage in several hypoxia-tolerant organisms under hypoxia. In addition, over the years, the idea of an assured decrease in ROS formation under hypoxia was challenged. Instead, several findings indicate that the production of ROS actually increases in response to hypoxia. Recently, it became possible to provide a comprehensive explanation for the induction of antioxidant enzymes under hypoxia. The supporting evidence and the limitations of the POS idea are extensively explored in this review as we discuss results from research on estivation and situations of low oxygen stress, such as hypoxia, freezing exposure, severe dehydration, and air exposure of water-breathing animals. We propose that, under some level of oxygen deprivation, ROS are overproduced and induce changes leading to hypoxic biochemical responses. These responses would occur mainly through the activation of specific transcription factors (FoxO, Nrf2, HIF-1, NF-κB, and p53) and post translational mechanisms, both mechanisms leading to enhanced antioxidant defenses. Moreover, reactive nitrogen species are candidate modulators of ROS generation in this scenario. We conclude by drawing out the future perspectives in this field of research, and how advances in the knowledge of the mechanisms involved in the POS strategy will offer new and innovative study scenarios of biological and physiological cellular responses to environmental stress.

Role of redox metabolism for adaptation of aquatic animals to drastic changes in oxygen availability.

Large changes in oxygen availability in aquatic environments, ranging from anoxia through to hyperoxia, can lead to corresponding wide variation in the production of reactive oxygen species (ROS) by animals with aquatic respiration. Therefore, animals living in marine, estuarine and freshwater environments have developed efficient antioxidant defenses to minimize oxidative stress and to regulate the cellular actions of ROS. Changes in oxygen levels may lead to bursts of ROS generation that can be particularly harmful. This situation is commonly experienced by aquatic animals during abrupt transitions from periods of hypoxia/anoxia back to oxygenated conditions (e.g. intertidal cycles). The strategies developed differ significantly among aquatic species and are (i) improvement of their endogenous antioxidant system under hyperoxia (that leads to increased ROS formation) or other similar ROS-related stresses, (ii) increase in antioxidant levels when displaying higher metabolic rates, (iii) presence of constitutively high levels of antioxidants, that attenuates oxidative stress derived from fluctuations in oxygen availability, or (iv) increase in the activity of antioxidant enzymes (and/or the levels of their mRNAs) during hypometabolic states associated with anoxia/hypoxia. This enhancement of the antioxidant system - coined over a decade ago as "preparation for oxidative stress" - controls the possible harmful effects of increased ROS formation during hypoxia/reoxygenation. The present article proposes a novel explanation for the biochemical and molecular mechanisms involved in this phenomenon that could be triggered by hypoxia-induced ROS formation. We also discuss the connections among oxygen sensing, oxidative damage and regulation of the endogenous antioxidant defense apparatus in animals adapted to many natural or man-made challenges of the aquatic environment.

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.

Role of catalase on the hypoxia/reoxygenation stress in the hypoxia-tolerant Nile tilapia.

The specific contribution of each antioxidant enzyme to protection against the reoxygenation-associated oxidative stress after periods of hypoxia is not well understood. We assessed the physiological role of catalase during posthypoxic reoxygenation by the combination of two approaches. First, catalase activity of Nile tilapias (Oreochromis niloticus) was 90% suppressed by intraperitoneal injection of 3-amino-1,2,4-triazole (ATZ, 1g/kg). In ATZ-injected fish, liver GSH levels, oxidative stress markers, and activities of other antioxidant enzymes remained unchanged. Second, animals with depleted catalase activity (or those saline-injected) were subjected to a cycle of severe hypoxia (dissolved O(2) = 0.28 mg/l for 3 h) followed by reoxygenation (0.5 to 24 h). Hypoxia did not induce changes in the above-mentioned parameters, either in saline- or in ATZ-injected animals. Reoxygenation increased superoxide dismutase activity in saline-injected fish, whose levels were similar to ATZ-injected animals. The activities of glutathione S-transferase, selenium-dependent glutathione peroxidase, and total-GPX and the levels of GSH-eq, GSSG, and thiobarbituric acid reactive substances remained unchanged during reoxygenation in both saline- and ATZ-injected fish. The GSSG/GSH-eq ratio in ATZ-injected fish increased at 30 min of reoxygenation compared with saline-injected ones. Reoxygenation also increased carbonyl protein levels in saline-injected fish, whose levels were similar to the ATZ-injected group. Our work shows that inhibition of liver tilapia catalase causes a redox imbalance during reoxygenation, which is insufficient to induce further oxidative stress. This indicates the relevance of hepatic catalase for hypoxia/reoxygenation stress in tilapia fish.

Cipura paludosa attenuates long-term behavioral deficits in rats exposed to methylmercury during early development.

In the present study, we evaluated the effects of the ethanolic extract (EE) of Cipura paludosa on locomotor, and anxiety- and depression-like behaviors of adult rats exposed to MeHg during early development. Additionally, the antioxidant enzymes catalase (CAT) and selenium-glutathione peroxidase (Se-GPx) were measured in cortical, hippocampal, and cerebellar tissues. Pregnant Wistar rats were treated by gavage with a single dose of MeHg (8 mg/kg) on gestational day 15, the developmental stage critical for cortical neuron proliferation. Moreover, prenatal MeHg exposure inhibited CAT and Se-GPx in the cortex and cerebellum. Chronic treatment with the EE of C. paludosa attenuated these emotional and antioxidant deficits induced by prenatal MeHg toxic exposure. This study provides novel evidence that developmental exposure to MeHg can affect not only cognitive functions but also locomotor, and anxiety- and depression-like behaviors.

Oxidative stress response in Paracoccidioides brasiliensis: assessing catalase and cytochrome c peroxidase.

Paracoccidioides brasiliensis is a dimorphic fungus that infects humans and establishes infection in the yeast form. We are interested in the mechanisms this fungus uses to evade the human immune system, and in its survival strategies within infected host cells. Reactive oxygen species play an important role in host defence, but are detoxified by pathogen-derived antioxidant enzymes to prevent oxidative damage. The transcriptional and post-transcriptional regulation of P. brasiliensis catalase and cytochrome-c peroxidase (CCP) antioxidant enzymes upon culture treatment with hydrogen peroxide (H(2)O(2)) is described. High H(2)O(2) concentrations (up to 100 mm) still permitted 70-100% survival of exponential and stationary phase yeast cells, though stationary phase cells were consistently more resistant. P. brasiliensis has both cytosolic and peroxisomal catalase isoenzymes and a single cytochrome-c peroxidase. High-dose treatments with H(2)O(2) led to an early increase in total catalase and CCP enzymatic activities, indicative of post-transcriptional regulation. The expression levels of the catalase genes increased three to fourfold when the cells were treated with 50 mm H(2)O(2) for 40 or 50 min. Lipid peroxidation, as assessed by the thiobarbituric acid method, was relatively low upon treatment with H(2)O(2), which was consistent with our results demonstrating that P. brasiliensis has a powerful antioxidant defence system enabling it to survive H(2)O(2)-mediated stress.

Early transcriptional response of Paracoccidioides brasiliensis upon internalization by murine macrophages.

Paracoccidioides brasiliensis, a thermal dimorphic fungus, is the etiologic agent of the most common systemic mycosis in Latin America, paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen being able to survive and replicate within the phagosome of nonactivated murine and human macrophages. This ability has been proposed to be crucial to the development of disease. Thus, P. brasiliensis may have evolved mechanisms that counteract the constraints imposed by phagocytic cells. By using cDNA microarray technology we evaluated the early transcriptional response of this fungus to the environment of peritoneal murine macrophages in order to shed light on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes analyzed, we identified 152 genes that were differentially transcribed. Intracellularly expressed genes were primarily associated with glucose and amino acid limitation, cell wall construction, and oxidative stress. For the first time, a comprehensive gene expression tool is used for the expression analysis of P. brasiliensis genes when interacting with macrophages. Overall, our data show a transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages which may lead to adaptation and consequent survival of this pathogen.

Oxidative stress response in Paracoccidioides brasiliensis.

Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.

Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.

Transcriptional profiles of the human pathogenic fungus Paracoccidioides brasiliensis in mycelium and yeast cells.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Overview and perspectives the transcriptome of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.