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Complete insect metamorphosis requires substantial metabolic and physiological adjustments. Although oxidative stress has been implicated in metamorphosis, details on redox metabolism during larva-to-pupa and pupa-to-adult remain scarce. This study explores redox metabolism during metamorphosis of a lepidopteran ( Chlosyne lacinia ), focusing on core metabolism, antioxidant systems and oxidative stress. The larva-to-pupa transition was characterized by increased lactate dehydrogenase and glutathione peroxidase (GPX) activities, coupled with depletion of reduced glutathione (GSH), high disulfide-to-total-glutathione ratio (GSSG/tGSH), and increased lipid peroxidation. As metamorphosis progressed, metabolic enzyme activities, citrate synthase and glucose 6-phosphate dehydrogenase increased, indicating heightened oxidative metabolism associated with adult development. Concurrently, GSH and GPX levels returned to larval levels and GSSG/tGSH reached its most reduced state right before adult emergence. Adult emergence was marked by a further increase in oxidative metabolism, accompanied by redox imbalance and enhanced antioxidant mechanisms. These findings highlight a fluctuation in redox balance throughout metamorphosis, with periods of oxidative eustress followed by compensatory antioxidant responses. This study is the first to identify concurrent changes in metabolism, antioxidants, redox balance and oxidative stress throughout metamorphosis. Our findings extend knowledge on redox metabolism adjustments and highlight redox adaptations and oxidative stress as natural components of complete insect metamorphosis.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The upregulation of endogenous antioxidants is a widespread phenomenon in animals that tolerate hypoxia/anoxia for extended periods. The identity of the mobilized antioxidant is often context-dependent and differs among species, tissues, and stresses. Thus, the contribution of individual antioxidants to the adaptation to oxygen deprivation remains elusive. This study investigated the role of glutathione (GSH) in the control of redox homeostasis under the stress of anoxia and reoxygenation in Helix aspersa , an animal model of anoxia tolerance. To do so, the total GSH (tGSH) pool was depleted with l-buthionine-(S, R)-sulfoximine (BSO) before exposing snails to anoxia for 6 h. Then, the concentration of GSH, glutathione disulfide (GSSG), and oxidative stress markers (TBARS and protein carbonyl) and the activity of antioxidant enzymes (catalase, glutathione peroxidase, glutathione transferase, glutathione reductase, and glucose 6-phosphate dehydrogenase) were measured in foot muscle and hepatopancreas. BSO alone induced tGSH depletion by 59-75%, but no other changes happened in other variables, except for foot GSSG. Anoxia elicited a 110-114% increase in glutathione peroxidase in the foot; no other changes occurred during anoxia. However, GSH depletion before anoxia increased the GSSG/tGSH ratio by 84-90% in both tissues, which returned to baseline levels during reoxygenation. Our findings indicate that glutathione is required to withstand the oxidative challenge induced by hypoxia and reoxygenation in land snails.

Preparation for oxidative stress (POS) is a widespread adaptive response to harsh environmental conditions, whose hallmark is the upregulation of antioxidants. In contrast to controlled laboratory settings, animals are exposed to multiple abiotic stressors under natural field conditions. Still, the interplay between different environmental factors in modulating redox metabolism in natural settings remains largely unexplored. Here, we aim to shed light on this topic by assessing changes in redox metabolism in the mussel Brachidontes solisianus naturally exposed to a tidal cycle. We compared the redox biochemical response of mussels under six different natural conditions in the field along two consecutive days. These conditions differ in terms of chronology, immersion/emersion, and solar radiation, but not in terms of temperature. Animals were collected after being exposed to air early morning (7:30), immersed during late morning and afternoon (8:45-15:30), and then exposed to air again late afternoon towards evening (17:45-21:25), in two days. Whole body homogenates were used to measure the activity of antioxidant (catalase, glutathione transferase and glutathione reductase) and metabolic (glucose 6-phosphate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and pyruvate kinase) enzymes, reduced (GSH) and disulfide (GSSG) glutathione levels, and oxidative stress markers (protein carbonyl and thiobarbituric acid reactive substances). Air and water temperature remained stable between 22.5 °C and 26 °C during both days. Global solar radiation (GSR) greatly differed between days, with a cumulative GSR of 15,381 kJ/m 2 for day 1 and 5,489 kJ/m 2 for day 2, whose peaks were 2,240 kJ/m 2 /h at 14:00 on day 1 and 952 kJ/m 2 /h at 12:00 on day 2. Compared with animals underwater, emersion during early morning did not elicit any alteration in redox biomarkers in both days. Air exposure for 4 h in the late afternoon towards evening caused oxidative damage to proteins and lipids and elicited GSH synthesis in animals that had been previously exposed to high GSR during the day. In the following day, when GSR was much lower, exposure to air under the same conditions (duration, time, and temperature) had no effect on any redox biomarker. These findings suggest that air exposure under low-intensity solar radiation is not sufficient to trigger POS in B. solisianus in its natural habitat. Thus, natural UV radiation is possibly a key environmental factor that combined to air exposure induces the POS-response to the stressful event of tidal variation in this coastal species., Competing Interests: Daniel C. Moreira is an Academic Editor for PeerJ., (©2023 Moreira et al.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The upregulation of endogenous antioxidants (i.e., preparation for oxidative stress, POS) is part of the biochemical responses underlying the adaptation of animals to adverse environments. Despite the phylogenetic diversity of animals in which POS has been described, most studies focus on animals under controlled laboratory conditions. To address this limitation, we have recently assessed the redox metabolism in the skeletal muscle of Proceratophrys cristiceps estivating under natural settings in the Caatinga. Here, we analyzed biochemical biomarkers in the muscle of another Caatinga species, Pleurodema diplolister , during the rainy (active) and dry (estivating frogs) seasons. We aimed to determine whether P. diplolister enhances its antioxidants during estivation under field conditions and to identify any effect of species on the biochemical responses of P. diplolister and P. cristiceps associated with estivation. To do so, we measured the activities of representative enzymes of intermediary metabolism and antioxidant systems, as well as glutathione and protein carbonyl levels, in the skeletal muscle of P. diplolister . Our findings revealed the suppression of oxidative metabolism and activation of antioxidant enzymes in estivating P. diplolister compared with active specimens. No changes in oxidative damage to proteins were observed and estivating P. diplolister had lower levels of disulfide glutathione (GSSG) and disulfide-to-total glutathione ratio (GSSG/tGSH) than those observed in active individuals. When data for P. diplolister and P. cristiceps were assembled and analyzed, significant effects of species were detected on the activities of metabolic enzymes (citrate synthase, isocitric dehydrogenase, malic enzyme, and creatine kinase) and antioxidant enzymes (catalase, glutathione peroxidase and glutathione transferase), as well as on GSSG/tGSH ratio. Such effects might underlie the physiological and behavioral differences between these two species that share the same microhabitat and survival strategy (i.e., to estivate) during the dry season. Despite some peculiarities, which reflect the physiological diversity of the mechanisms associated with estivation in the Brazilian Caatinga, both P. diplolister and P. cristiceps seem to balance the suppression of oxidative pathways, the maintenance of the capacity of oxygen-independent pathways, and the activation of endogenous antioxidants to preserve muscle function and be ready to resume activity whenever the unpredictable rainy period arrives., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moreira, Carvajalino-Fernández, Navas, de Carvalho and Hermes-Lima.)

Insect diapause shares many biochemical features with other states of metabolic depression, including the suppression of global metabolism, reorganization of metabolic pathways and improved stress resistance. However, little is known about the biochemical changes associated with the diapause phenotype in tropical insects. To investigate biochemical adaptations associated with tropical diapause, we measured the activities of metabolic and antioxidant enzymes, as well as glutathione levels, in the sunflower caterpillar Chlosyne lacinia at different times after initiation of diapause (<1, 20, 40, 60, and 120 days) and after arousal from diapause. Biochemical changes occurred early in diapausing animals, between the first 24 h and 20 days of diapause. Diapausing animals had reduced oxidative capacity associated with a decrease in the activities of peroxide-decomposing antioxidant enzymes. There was no sign of redox imbalance either during diapause or after recovery from diapause. Noteworthy, glutathione transferase and isocitrate dehydrogenase-NADP + activities sharply increased in diapausing animals and stand out as diapause-associated proteins. The upregulation of these two enzymes ultimately indicate the occurrence of Preparation for Oxidative Stress in the tropical diapause of C. lacinia., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Preparation for oxidative stress (POS), i.e., the buildup of endogenous antioxidants during metabolic depression or low oxygen stress conditions, has been observed in at least 8 animal phyla under controlled conditions in laboratory. Despite the expected implications on the endurance to extreme environments and ecosystem occupation, the extent to which POS occurs in animals under natural conditions remains unexplored. Therefore, we took advantage of the natural history of the Brazilian Caatinga's frog Proceratophrys cristiceps to investigate the modulation of endogenous antioxidants and redox balance in their skeletal muscle and to verify if POS occurs under natural conditions. Expectedly, estivating frogs had low levels of the oxidative metabolism enzymes. Citrate synthase and isocitrate dehydrogenase activities were 36% and 25% lower than those in active frogs respectively. We found an overall upregulation of antioxidants in estivating P. cristiceps. Reduced glutathione concentration was 61% higher in estivating frogs than that in active animals. During estivation the activities of the hydroperoxide detoxification enzymes catalase, glutathione peroxidase, and glutathione H 2 O 2 -peroxidase were 48%, 57%, and 78% greater than those during the rainy season. Moreover, estivating frogs had a 47% lower ratio of disulfide to total glutathione levels than active frogs. Our findings confirm the occurrence of 'preparation for oxidative stress' in naturally estivating frogs and paves the way for further research on the redox biology of animals under natural settings. Such approach might reveal biochemical strategies under ecologically relevant scenarios., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Preparation for oxidative stress (POS), i.e., the upregulation of endogenous antioxidants, is a widespread response of animals exposed to extreme conditions. This response has been described for more than 80 animal species belonging to eight phyla during hypometabolism or situations that limit oxygen availability. The pattern of the typical POS-response, in which a mild redox imbalance triggers antioxidant adjustments that results in increased tolerance to subsequent oxidative insults, roughly follows the curve of hormetic phenomena. A similar pattern has been reported for various animal species exposed to ultraviolet radiation (UVR) - these studies, on animals from six phyla, are discussed herein. In the light of the similarities in the redox-response of animals exposed to either oxygen restriction or UVR, we argue in this essay that UVR elicits a type of response that fits the POS theory. Exposure to UVR induces both reactive species formation and antioxidant adaptation, which is the essence of typical POS-responses. Thus, antioxidant response to UVR in animals can be categorized as a POS-type mechanism. Moreover, considering that animals are exposed to multiple stressors simultaneously in nature, this would represent an ecologically relevant process, by which one stressor (e.g., UV or ionizing radiation) may enhance the tolerance to other. We also discuss a possible role of low doses of ionizing radiation as inductor of POS-like responses in animals., (Copyright © 2019. Published by Elsevier Inc.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare there are no competing interests.

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

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.

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.

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., (© 2016 The International Union of Biochemistry and Molecular Biology.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)