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.

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.

Aging and retinoid X receptor agonists on masculinization of female Pomacea canaliculata, with a critical appraisal of imposex evaluation in the Ampullariidae.

Ampullariidae are unique among gastropods in that females normally show a primordium of the copulatory apparatus (CApp). The aims of this study were (a) to quantitatively evaluate the development and growth of the female CApp with age; (b) to compare the effects of RXR and PPARγ agonists in adult females of known age and (c) to explore the effect of masculinizing RXR agonists on the expression of RXR in the CApp. It was found that the CApp grows and develops with age. A significant increase in penile sheath length (PsL) and also in a developmental index (DI) was observed in 7-8 months old females, as compared with 4-5 months old ones. A reported endogenous agonist of RXR, 9-cis retinoic acid (9cis-RA), as well as two organotin compounds, tributyltin (TBT) and triphenyltin (TPT) which have been also reported to bind to RXR, were injected and its masculinizing effects were measured. Also, the effect of a PPARγ agonist, rosiglitazone, was studied. All studied RXR agonists, but not the PPARγ agonist, were effective in increasing PsL, penile length (PL) and DI. Finally, the expression of the RXR in the CApp was studied (Western blot) in control, TBT, TPT, and 9cis-RA treated females. A significantly increased expression of RXR was only observed after 9cis-RA treatment. It is concluded that (a) development and growth of the CApp is significantly affected by female age; (b) reported RXR agonists, but not a PPARγ agonist, cause female masculinization of young females. An appraisal of previous studies of female masculinization in the Ampullariidae has also been made and it is emphasized that the masculinizing effect of aging should be considered, particularly when interpreting field data.

Antioxidant and molecular chaperone defences during estivation and arousal in the South American apple snail Pomacea canaliculata.

The invasive Pomacea canaliculata estivates during periods of drought and should cope with harmful effects of reoxygenation during arousal. We studied thiobarbituric acid reactive substances (TBARS), enzymatic (superoxide dismutase, SOD and catalase, CAT) and non-enzymatic antioxidants (uric acid and reduced glutathione), and heat shock protein expression (Hsc70, Hsp70 and Hsp90) in (1) active control snails, (2) snails after 45 days of estivation, and (3) aroused snails 20 min and (4) 24 h after water exposure, in midgut gland, kidney and foot. Both kidney and foot (but not the midgut gland) showed a TBARS increase during estivation and a decrease after arousal. Tissue SOD and CAT did not change in any experimental groups. Uric acid increased during estivation in all tissues, and it decreased after arousal in the kidney. Allantoin, the oxidation product of uric acid, remained constant in the midgut gland but it decreased in the kidney until 20 min after arousal; however, allantoin levels rose in both kidney and foot 24 h after arousal. Reduced glutathione decreased during estivation and arousal, in both midgut gland and kidney, and it remained constant in the foot. Hsc70 and Hsp70 kidney levels were stable during the activity-estivation cycle and Hsp90 expression decreases during estivation and recovers in the early arousal. In foot, the expression of Hsp70 and Hsp90 was high during activity and estivation periods and disminished after arousal. Results indicate that a panoply of antioxidant and molecular chaperone defences may be involved during the activity-estivation cycle in this freshwater gastropod.

Short-Term Estivation and Hibernation Induce Changes in the Blood and Circulating Hemocytes of the Apple Snail Pomacea canaliculata.

States of natural dormancy include estivation and hibernation. Ampullariids are exemplary because they undergo estivation when deprived of water or hibernation when exposed to very low temperatures. Regardless of the condition, ampullariids show increased endogenous antioxidant defenses, anticipating the expected respiratory burst during reoxygenation after reactivation, known as "Preparation for Oxidative Stress (POS)". In this work, we tested the POS hypothesis for changes in the blood and hemocytes of the bimodal breather Pomacea canaliculata (Ampullariidae) induced at experimental estivation and hibernation. We described respiratory (hemocyanin, proteins, lactate), antioxidant (GSH, uric acid, SOD, CAT, GST), and immunological (hemocyte levels, ROS production) parameters. We showed that, although the protein level remains unchanged in all experimental groups, hemocyanin increases in response to estivation. Furthermore, lactate remains unchanged in challenged snails, suggesting an aerobic metabolism during short-term challenges. Blood uric acid increases during estivation and arousal from estivation or hibernation, supporting the previously proposed antioxidant role. Regarding hemocytes, we showed that the total population increases with all challenges, and granulocytes increase during hibernation. We further showed that hibernation affects ROS production by hemocytes, possibly through mitochondrial inhibition. This study contributed to the knowledge of the adaptive strategies of ampullariids to tolerate adverse environmental conditions.

Changes in the oxidative status and damage by non-essential elements in the digestive gland of the gastropod Pomacea canaliculata.

The freshwater gastropod Pomacea canaliculata fulfills the ideal conditions of a bioindicator species since its digestive gland bioconcentrates elements toxic for human and ecosystems health. The aim of this work was to study the balance between production of free radicals and antioxidant defenses, and the generation of oxidative damage in the digestive gland of this mollusk after exposure (96 h) to three elements with differential affinities for functional biological groups: mercury (5.5 µg/L of Hg+2 as HgCl2), arsenic [500 µg/L of (AsO4)-3 as Na3AsO47H2O], or uranium [700 µg/L of (UO2)+2 as UO2(CH2COOH)2]. Bioconcentration factors of Hg, As, and U were 25, 23, and 53, respectively. Snails exhibited a sustained increase of reactive species (RS), and protein and lipid damage. Lipid radicals increased between 72 and 96 h, respectively, in snails exposed to U and Hg while this parameter changed early (24 h) in As- exposed snails. Snails showed protein damage, reaching maximum values at different endpoints. This redox disbalance was partially compensated by non-enzymatic antioxidant defenses α-tocopherol (α-T), ß-carotene (ß-C), uric acid, metallothionein (MTs). Snails consumed α-T and ß-C in an element-dependent manner. The digestive gland consumed rapidly uric acid and this molecule was not recovered at 96 h. Digestive gland showed a significant increase in MTs after elemental exposure at different endpoints. The enzymatic antioxidant defenses, represented by the catalase and glutathione-S-transferase activities, seems to be not necessary for the early stages of the oxidative process by metals. This work is the first attempt to elucidate cellular mechanisms involved in the tolerance of this gastropod to non-essential elements. The bioconcentration factors and changes in the oxidative status and damage confirm that this species can be used as a bioindicator species of metal pollution in freshwater bodies.

Antioxidant Responses Induced by Short-Term Activity-Estivation-Arousal Cycle in Pomacea canaliculata.

Long-term estivation (45 days) in the apple snail Pomacea canaliculata induces an increase of non-enzymatic antioxidants, such as uric acid and reduced glutathione (GSH), which constitutes an alternative to the adaptive physiological strategy of preparation for oxidative stress (POS). Here, we studied markers of oxidative stress damage, uric acid levels, and non-enzymatic antioxidant capacity, enzymatic antioxidant defenses, such as superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and transcription factors expression [forkhead box protein O (FOXO), hypoxia-inducible factor-1 alpha (HIF1α), and nuclear factor erythroid 2-related factor 2 (Nrf2)] in control active animals, 7-day estivating and aroused snails, in digestive gland, gill, and lung tissue samples. In the digestive gland, SOD and CAT activities significantly increased after estivation and decreased during arousal. Meanwhile, GST activity decreased significantly during the activity-estivation-arousal cycle. Gill CAT activity increased significantly at 7 days of estivation, and it decreased during arousal. In the lung, the CAT activity level increased significantly during the cycle. FOXO upregulation was observed in the studied tissues, decreasing its expression only in the gill of aroused animals during the cycle. HIF1α and Nrf2 transcription factors decreased their expression during estivation in the gill, while in the lung and the digestive gland, both transcription factors did not show significant changes. Our results showed that the short-term estivation induced oxidative stress in different tissues of P. canaliculata thereby increasing overall antioxidant enzymes activity and highlighting the role of FOXO regulation as a possible underlying mechanism of the POS strategy.

Evaluation of female masculinization in Pomacea canaliculata (Caenogastropoda, Ampullariidae) induced by tributyltin, heavy metals, and uranium in culture water.

All female Pomacea canaliculata develop a small, male-like copulatory apparatus a few days after birth, which growths slowly until sexual maturity, and even further in older age. Previous studies have found trace elements like mercury (Hg), arsenic (As) and uranium (U) in tap water used for snail culture, and that these elements were accumulated in snail tissues. Here, we test whether the presence of these metals at maximum allowed concentrations (Environmental Protection Agency - EPA) in aquarium water could affect the development of the copulatory apparatus in mature females. Females of different ages were used as controls, grown in reconstituted metal-free water with or without the addition of Hg, As and U, as well as tributyltin (TBT), a compound used as masculinizing agent. Six and seven months old females cultured in tap water showed a longer penis and penile sheath, and a greater overall development of the copulatory apparatus, measured by an index (DI), as compared with same-age females cultured in reconstituted water. Moreover, when females were exposed to Hg, As or U at the maximum contaminant levels for human consumption allowed by EPA regulations, there was no further development of the copulatory apparatus, while there was a clearly positive effect in TBT-exposed females. This study confirms the masculinizing effect of organotin compounds on female copulatory apparatus and discusses the usefulness of the development of these organs as a bioindicator of environmental pollution.

Uric acid deposits and estivation in the invasive apple-snail, Pomacea canaliculata.

The physiological ability to estivate is relevant for the maintenance of population size in the invasive Pomacea canaliculata. However, tissue reoxygenation during arousal from estivation poses the problem of acute oxidative stress. Uric acid is a potent antioxidant in several systems and it is stored in specialized tissues of P. canaliculata. Changes in tissue concentration of thiobarbituric acid reactive substances (TBARS), uric acid and allantoin were measured during estivation and arousal in P. canaliculata. Both TBARS and uric acid increased two-fold during 45 days estivation, probably as a consequence of concomitant oxyradical production during uric acid synthesis by xanthine oxidase. However, after arousal was induced, uric acid and TBARS dropped to or near baseline levels within 20 min and remained low up to 24h after arousal induction, while the urate oxidation product allantoin continuously rose to a maximum at 24h after induction, indicating the participation of uric acid as an antioxidant during reoxygenation. Neither uric acid nor allantoin was detected in the excreta during this 24h period. Urate oxidase activity was also found in organs of active snails, but activity shut down during estivation and only a partial and sustained recovery was observed in the midgut gland.

Monitoring of SARS-CoV-2 RNA in wastewater as an epidemiological surveillance tool in Mendoza, Argentina.

Wastewater-based epidemiology (WBE) is an emerging tool that gives temporal and spatial information on a population's health status. Here, we report the epidemiological dynamics of a population of ~1.2 million residents in the metropolitan region of Mendoza province, Argentina, within the period July 2020 to January 2021. We combined the use of WBE of two wastewater treatment plants with epidemiological surveillance of the corresponding populations. We applied two viral concentration methods (polyethylene glycol precipitation and aluminum-based adsorption-flocculation) and RNA isolation methods in each wastewater sample to increase the possibility of detection and quantification of nucleocapsid markers (N1 and N2) of SARS-CoV-2 by RT-qPCR. Overall, our results allowed us to trace the rise, exponential growth, plateau, and fall of SARS-CoV-2 infections for 26 weeks. Individual analysis for each wastewater treatment plant showed a positive correlation between the viral load of SARS-CoV-2 genetic markers and COVID-19 cases that were diagnosed per week. Our findings indicate that WBE is a useful epidemiological indicator to anticipate the increase in COVID-19 cases and monitor the advance of the pandemic and different waves of infections.

Tolerance to hypometabolism and arousal induced by hibernation in the apple snail Pomacea canaliculata (Caenogastropoda, Ampullariidae).

Pomacea canaliculata may serve as a model organism for comparative studies of oxidative damage and antioxidant defenses in active, hibernating and arousing snails. Oxidative damage (as TBARS), free radical scavenging capacity (as ABTS+ oxidation), uric acid (UA) and glutathione (GSH) concentrations, activities of superoxide dismutase (SOD) and catalase (CAT), and the protein expression levels of heat shock proteins (Hsp70, Hsc70, Hsp90) were studied in digestive gland, kidney and foot. Tissue TBARS of hibernating snails (45days) was higher than active snails. Hibernation produced an increase of ABTS+ in digestive gland, probably because of the sustained antioxidant defenses (UA and/or GSH and SOD levels). Kidney protection during the activity-hibernation cycle seemed provided by increased UA concentrations. TBARS in the foot remained high 30min after arousal with no changes in ABTS+, but this tissue increased ABTS+ oxidation at 24h to expenses increased UA and decreased GSH levels, and with no changes in SOD and CAT activities. The level of Hsp70 in kidney showed no changes throughout the activity-hibernation cycle but it increased in the foot after hibernation. The tissue levels of Hsp90 in snails hibernating were higher than active snails and returned to baseline 24h after arousal. Results showed that chronic cooling produces a significant oxidative damage in three studied tissues and that these tissue damages are overcome quickly (between 30min to 24h) with fluctuations in different antioxidant defenses (UA, GSH, CAT) and heat shock proteins (Hsp70 and Hsp90).

Proregenerative Microenvironment Triggered by Donor Mesenchymal Stem Cells Preserves Renal Function and Structure in Mice with Severe Diabetes Mellitus.

The aim of our work was to evaluate, in an animal model of severe diabetes mellitus, the effect of mesenchymal stem cells (MSCs) administration on diabetic nephropathy (DN) progression. After diabetes induction, one group of mice received the vehicle (DM) and other group received a single dose of MSCs (DM + MSCs). DM + MSCs mice showed a significant improvement in functional parameters of the kidney compared with untreated mice. While DM mice presented marked histopathological changes characteristics of advanced stages of DN (fibrosis, glomerulosclerosis, glomerular basement membrane thickening, capillary occlusion, decreased podocyte density, and effacement of foot processes), DM + MSCs mice showed only slight tubular dilatation. The renoprotection was not associated with an improvement in diabetic condition and very low number of donor cells was found in the kidney of DM + MSCs mice, suggesting that renoprotection could be mediated by paracrine effects. Indeed, DM + MSC mice presented increased renal proliferation index, decreased renal apoptotic index and the restoration of proregenerative factors, and anti-inflammatory cytokines levels. Moreover, macrophage infiltration and oxidative stress damage were also reduced in DM + MSCs mice. Our data demonstrate that MSC administration triggers a proregenerative microenvironment in DN kidney, which allows the preservation of the renal function even if diabetes was uncorrected.

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.

Imposex and novel mechanisms of reproductive failure induced by tributyltin (TBT) in the freshwater snail Pomacea canaliculata.

The effects of tributyltin (TBT) on mortality and reproduction were studied in the invasive snail Pomacea canaliculata. The nominal median lethal concentration (LC50) was 9 µg TBT/L, after 28 d. The nominal no-observed effect on lethality concentration (NOECL) was 6 µg TBT/L after the same period. Male-female couples and females that had been group-mated but were isolated from males during the experiment (isolated females) were exposed (for 28 d) to either 0 µg/L or 6 µg/L of TBT (nominal NOECL-exposed). Copulation and oviposition frequencies, egg clutch mass, and percentage of egg fertility were recorded. Gonads (both sexes) and the seminal receptacle (females) were studied histologically at the end of the experiment. A significant decrease in copulation frequency was observed in mated-exposed females. Exposure also decreased oviposition frequency of mated-exposed and isolated-exposed females, but only the latter reached significance. No differences in either egg clutch mass or percentage of fertility were observed at first oviposition, but both parameters were drastically reduced in subsequent egg clutches of exposed females. No histological alterations were observed in gonads of TBT-exposed animals; however, sperm storage in the seminal receptacle was drastically decreased in exposed females. Imposex but no oviductal obstruction was observed in all exposed females. It is concluded that TBT induces reproductive failure in P. canaliculata by decreasing copulation frequency and by severely affecting sperm storage by the female.