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The widely used insecticide chlorpyrifos (CP) is known to inhibit acetylcholinesterase (AChE) activity attributed to result in various neurological disorders and acetylcholine-dependent organ functions including heart, skeletal muscle, lung, gastrointestinal tract, and central nervous systems. Enzyme reactivators, such as oximes, are known to restore AChE activity and mitigate adverse effects. The identification of compounds that reactivate AChE constitute agents with important therapeutic beneficial effects in cases of pesticide poisoning. However, the screening of novel drugs using traditional models may raise ethical concerns. This study aimed to investigate the potential of Drosophila melanogaster as a model organism for screening AChE reactivators, with a focus on organophosphate poisoning. The efficacy of several oximes, including pralidoxime, trimedoxime, obidoxime, methoxime, HI-6, K027, and K048, against CP-induced AChE activity inhibition in D. melanogaster was determined in silico , in vitro , and in vivo experiments. Molecular docking studies indicated a strong interaction between studied oximes and the active-site gorge of AChE. Data showed that selected oximes (100 μM) are effective in the reactivation of AChE inhibited by CP (10 μM) in vitro . Finally, in vivo investigations demonstrated that selected oximes, pralidoxime and K048 (1.5 ppm), reversed the locomotor deficits, inhibition of AChE activity as well as lowered the mortality rates induced by CP (0.75 ppm). Our findings contribute to utilization of D. melanogaster as a robust model for determination of actions of identified new AChE inhibitory agents with more effective therapeutic properties that those currently in use in the clinical practice in treatment of AChE associated disorders.

REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research., Competing Interests: Declaration of competing interest The authors declare no conflict of interests, (Copyright © 2024 Elsevier Inc. All rights reserved.)

Copper (Cu) is a naturally occurring metal with essential micronutrient properties. However, this metal might also pose increased adverse environmental and health risks due to industrial and agricultural activities. In Brazil, the maximum allowable concentration of Cu in drinking water is 2 mg/L. Despite this standard, the impact of such concentrations on aquatic organisms remains unexplored. This study aimed to evaluate the toxicity of CuSO 4 using larval zebrafish at environmentally relevant concentrations. Zebrafish ( Danio rerio ) larvae at 72 hr post-fertilization (hpf) were exposed to nominal CuSO 4 concentrations ranging from 0.16 to 48 mg/L to determine the median lethal concentration (LC 50 ), established at 8.4 mg/L. Subsequently, non-lethal concentrations of 0.16, 0.32, or 1.6 mg/L were selected for assessing CuSO 4 -induced toxicity. Morphological parameters, including body length, yolk sac area, and swim bladder area, were adversely affected by CuSO 4 exposure, particularly at 1.6 mg/L (3.31 mm ±0.1, 0.192 mm 2 ±0.01, and 0.01 mm 2 ±0.05, respectively). In contrast, the control group exhibited values of 3.62 mm ±0.09, 0.136 mm 2 ±0.013, and 0.3 mm 2 ±0.06, respectively. Behavioral assays demonstrated impairments in escape response and swimming capacity, accompanied by increased levels of reactive oxygen species (ROS) and lipid peroxidation. In addition, decreased levels of non-protein thiols and reduced cellular viability were noted. Data demonstrated that exposure to CuSO 4 at similar concentrations as those permitted in Brazil for Cu adversely altered morphological, biochemical, and behavioral endpoints in zebrafish larvae. This study suggests that the permissible Cu concentrations in Brazil need to be reevaluated, given the potential enhanced adverse health risks of exposure to environmental metal contamination.

Huntington's disease (HD) is a neurodegenerative genetic disorder characterized by motor, psychiatric, cognitive, and peripheral symptoms without effective therapy. Evidence suggests that lifestyle factors can modulate disease onset and progression, and environmental enrichment (EE) has emerged as a potential approach to mitigate the progression and severity of neurodegenerative processes. Wild-type (WT) and yeast artificial chromosome (YAC) 128 mice were exposed to different EE conditions. Animals from cohort 1 were exposed to EE between postnatal days 21 and 60, and animals from cohort 2 were exposed to EE between postnatal days 60 and 120. Motor and non-motor behavioral tests were employed to evaluate the effects of EE on HD progression. Monoamine levels, hippocampal cell proliferation, neuronal differentiation, and dendritic arborization were also assessed. Here we show that EE had an antidepressant-like effect and slowed the progression of motor deficits in HD mice. It also reduced monoamine levels, which correlated with better motor performance, particularly in the striatum. EE also modulated neuronal differentiation in the YAC128 hippocampus. These results confirm that EE can impact behavior, hippocampal neuroplasticity, and monoamine levels in YAC128 mice, suggesting this could be a therapeutic strategy to modulate neuroplasticity deficits in HD. However, further research is needed to fully understand EE's mechanisms and long-term effects as an adjuvant therapy for this debilitating condition.

The association between chronic kidney disease (CKD) and pulmonary pathophysiological changes is well stablished. Nevertheless, the effects of aerobic exercise (AE) on lungs of CKD need further clarification. Thus, Swiss mice were divided in control, AE, CKD, and CKD + AE groups. CKD was induced by 0.2% adenine intake during 8 weeks (4 weeks of CKD induction and 4 weeks of AE). AE consisted in running on treadmill, at moderate intensity, 30 min/day, 5 days/week, during 4 weeks. Twenty-four hours after the last training day, functional capacity test was performed, and 48 h after the test, mice were euthanized. CKD mice showed a significant increase in urine output, serum urea, and creatinine concentrations, and decreased body weight and urine density, besides oxidative damage (p = 0.044), edema area (p < 0.001), leukocyte infiltration (p = 0.040), and collagen area in lung tissue (p = 0.004). AE resulted in an increase of distance traveled (p = 0.049) and maximum speed (p = 0.046), increased activity of catalase (p = 0.031) and glutathione peroxidase (p = 0.048) in lungs, increased levels of nitric oxide (NOx) in serum (p = 0.001) and bronchoalveolar lavage fluid (p = 0.047), and decreased kidney histological injury (p = 0.018) of CKD mice. However, AE also increased oxidative damage (p = 0.003) and did not change collagen content or perivascular edema in lungs (p > 0.05) of CKD mice. Therefore, AE attenuated kidney injury and improved antioxidants defenses in lungs. Despite no significant changes in pulmonary damage, AE significantly improved physical performance in CKD mice., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cholecalciferol deficiency has been associated with stress-related psychiatric disorders, particularly depression. Therefore, the present study investigated the antidepressant-like effect of cholecalciferol in female mice and the possible role of the serotonergic system in this response. The ability of cholecalciferol to elicit an antidepressant-like effect and to modulate serotonin levels in the hippocampus and prefrontal cortex of mice subjected to chronic unpredictable stress (CUS) was also investigated. The administration of cholecalciferol (2.5, 7.5, and 25 µg/kg, p.o.) for 7 days, similar to fluoxetine (10 mg/kg, p.o., serotonin reuptake inhibitor), reduced the immobility time in the tail suspension test, without altering the locomotor performance in the open-field test. Moreover, the administration of p-chlorophenylalanine methyl ester (PCPA - 100 mg/kg, i.p., for 4 days, a selective inhibitor of tryptophan hydroxylase, involved in the serotonin synthesis) abolished the antidepressant-like effect of cholecalciferol and fluoxetine in the tail suspension test, demonstrating the involvement of serotonergic system. Additionally, CUS protocol (21 days) induced depressive-like behavior in the tail suspension test and decreased serotonin levels in the prefrontal cortex and hippocampus of mice. Conversely, the administration of cholecalciferol and fluoxetine in the last 7 days of CUS protocol completely abolished the stress-induced depressive-like phenotype. Cholecalciferol was also effective to abrogate CUS-induced reduction on serotonin levels in the prefrontal cortex, but not in the hippocampus. Our results indicate that cholecalciferol has an antidepressant-like effect in mice by modulating the serotonergic system and support the assumption that cholecalciferol may have beneficial effects for the management of depression., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We investigated whether dietary supplementation with Aurantiochytrium sp. meal, a DHA-rich source (docosahexaenoic acid, 22: 6 n-3), fed during long-term exposure to cold-suboptimal temperature (22 °C, P1), followed by short-term exposure to higher temperatures (28 °C, P2, and 33 °C, P3), would promote oxidative damage in Nile tilapia (Oreochromis niloticus). Two supplementation levels were tested: 1.0 g 100 g -1 (D1) and 4.0 g 100 g -1 (D4). A control diet, without the additive (D0, 0 g 100 g -1 ), and a positive control diet supplemented with cod liver oil (CLO) were also tested. The concentrations of DHA and total n-3 PUFAs in the CLO diet were similar to those found in diets D1 and D4, respectively. The parameters analyzed included hemoglobin (Hb), the antioxidant enzymes catalase, glutathione peroxidase, total glutathione, non-protein thiols, and the oxidative markers protein carbonyl and erythrocyte DNA damage. Nile tilapia did not present differences in Hb content, regardless of diet composition, but the temperature increase (P1 to P2) led to a higher Hb content. Likewise, the temperature increases promoted alterations in all antioxidant enzymes. The dietary supplementation with 1.0 g 100 g -1 Aurantiochytrium sp. meal after P1 caused minor DNA damage in Nile tilapia, demonstrating that the additive can safely be included in winter diets, despite its high DHA concentration., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Methylglyoxal (MGO) is an endogenous toxin, mainly produced as a by-product of glycolysis that has been associated to aging, Alzheimer's disease, and inflammation. Cell culture studies reported that MGO could impair the glyoxalase, thioredoxin, and glutathione systems. Thus, we investigated the effect of in vivo MGO administration on these systems, but no major changes were observed in the glyoxalase, thioredoxin, and glutathione systems, as evaluated in the prefrontal cortex and the hippocampus of mice. A previous study from our group indicated that MGO administration produced learning/memory deficits and depression-like behavior. Confirming these findings, the tail suspension test indicated that MGO treatment for 7 days leads to depression-like behavior in three different mice strains. MGO treatment for 12 days induced working memory impairment, as evaluated in the Y maze spontaneous alternation test, which was paralleled by low dopamine and serotonin levels in the cerebral cortex. Increased DARPP32 Thr75/Thr34 phosphorylation ratio was observed, suggesting a suppression of phosphatase 1 inhibition, which may be involved in behavioral responses to MGO. Co-treatment with a dopamine/noradrenaline reuptake inhibitor (bupropion, 10 mg/kg, p.o.) reversed the depression-like behavior and working memory impairment and restored the serotonin and dopamine levels in the cerebral cortex. Overall, the cerebral cortex monoaminergic system appears to be a preferential target of MGO toxicity, a new potential therapeutic target that remains to be addressed.

Recent evidence suggests that young rodents submitted to high fructose (FRU) diet develop metabolic, and cognitive dysfunctions. However, it remains unclear whether these detrimental effects of FRU intake can also be observed in middle-aged mice. Nine months-old C57BL/6 female mice were fed with water (Control) or 10% FRU in drinking water during 12 weeks. After that, metabolic, and neurochemical alterations were evaluated, focusing on neurotransmitters, and antioxidant defenses. Behavioral parameters related to motor activity, memory, anxiety, and depression were also evaluated. Mice consuming FRU diet displayed increased water, and caloric intake, resulting in weight gain, which was partially compensated due to decreased food pellet intake. FRU fed animals displayed increased plasma glucose, and cholesterol levels, which was not observed in overnight-fasted animals. Superoxide dismutase (SOD), and catalase (CAT) activities were markedly decreased in the prefrontal cortex of animals receiving FRU diet, while glutathione peroxidase (GPx) slightly increased. Liver (lower GPx), striatum (higher SOD and lower CAT), and hippocampus (no changes) were less impacted. No changes were observed in glutathione reductase, and thioredoxin reductase activities, two ancillary enzymes for peroxide detoxification. FRU intake did not alter serotonin, dopamine, and norepinephrine levels in the hippocampus, prefrontal cortex, and striatum. No significant alterations were observed in working, and short-term spatial memory; and in anxiety- and depressive-like behaviors in animals treated with FRU. Increased locomotor activity was observed in FRU-fed middle-aged mice, as evaluated in the open field, elevated plus-maze, Y maze, and object location tasks. Overall, these results demonstrate that high FRU consumption can disturb antioxidant defenses, and increase locomotor activity in middle-aged mice, open the opportunity for further studies to address the underlying mechanisms related to these findings.

Processed meats are classified by the International Agency for Research on Cancer as category 1 because their consumption increase the incidence of colorectal and stomach cancers. Meat processing widely employs nitrite and sorbate as preservatives. When these preservatives are concomitantly used in non-compliant processes, they may react and produce the mutagen 2-methyl-1,4-dinitro-pyrrole (DNMP). This study aimed to evaluate the ability of different bacteria isolated from food matrices to biodegrade DNMP in in vitro reactions and in a processed meat model. A possible mechanism of biodegradation was also tested. In vitro experiments were performed in two steps. In the first one, only one strain out of 13 different species did not interact with DNMP. In the following step, an empirical conversion factor was calculated to assess the conversion of DNMP to 4-amino-2-methyl-1-nitro-pyrrole by the strains. The most efficient strains were Staphylococcus xylosus LYOCARNI SXH-01, Lactobacillus fermentum LB-UFSC 0017, and Lactobacillus casei LB-UFSC 0019, which yielded conversion factors of 0.62, 0.60, and 0.43, respectively. Thus, such strains were individually added to the processed meat model and completely degraded the DNMP. Moreover, S. xylosus degraded DNMP in less than 30 min. The enzymatic mechanism was evaluated using its cell-free extract. It showed that, in the aerobic system, reduction rates were 30.321 and 22.411 nmol/mg of protein/min using NADH and NADPH, respectively. A DNMP reductase was assigned to the extract and a potential presence of an oxygen insensitive nitroreductase type I B was considered. Thus, biotechnological processes may be an efficient strategy to eliminate the DNMP from meat products and to increase food safety., 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 Ltd. All rights reserved.)

A previous study demonstrated that glutathione (GSH) produces specific antidepressant-like effect in the forced swimming test (FST), a predictive test of antidepressant activity. The present study investigated the involvement of multiple cellular targets implicated in the antidepressant-like effect of GSH in the FST. The antidepressant-like effect of GSH (300 nmol/site, icv) lasted up to 3 h when mice were submitted to FST. The central administration of oxidized GSH (GSSG, 3-300 nmol/site) did not alter the behavior of mice submitted to the FST. Furthermore, the combined treatment of sub-effective doses of GSH (100 nmol/site, icv) with a sub-effective dose of classical antidepressants (fluoxetine 10 mg/kg, and imipramine 5 mg/kg, ip) presented synergistic effect by decreasing the immobility time in the FST. The antidepressant-like effect of GSH was abolished by prazosin (1 mg/kg, ip, α 1 -adrenoceptor antagonist), baclofen (1 mg/kg, ip, GABAB receptor agonist), bicuculline (1 mg/kg, ip, GABAA receptor antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/site, icv, NO donor), but not by yohimbine (1 mg/kg, ip, α 2 -adrenoceptor antagonist). The NMDA receptor antagonists, MK-801(0.001 mg/kg, ip) or GMP (0.5 mg/kg, ip), potentiated the effect of a sub-effective dose of GSH in the FST. These results suggest that the antidepressant-like effect induced by GSH is connected to the activation of α1 adrenergic and GABAA receptors, as well as the inhibition of GABAB and NMDA receptors and NO biosyntesis. We speculate that redox-mediated signaling on the extracelular portion of cell membrane receptors would be a common mechanism of action of GSH., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Exposure to fungicide ziram (zinc dimethyldithiocarbamate) has been associated with increased incidence of Parkinson's disease (PD). We recently demonstrated that the intranasal (i.n.) administration of sodium dimethyldithiocarbamate (NaDMDC, a more soluble salt than ziram) induces PD-like behavioral and neurochemical alterations in mice. We now investigated the putative neuroprotective effects of melatonin on behavioral dificits and neurochemical alterations induced by i.n. NaDMDC. Melatonin treatment (3, 10 or 30 mg/kg, i.p.) was given 1 h before NaDMDC administration (1 mg/nostril) during 4 consecutive days and we evaluated early (up to 7 days) and late (up to 35 days) NaDMDC-induced behavioral and neurochemical alterations. Melatonin treatment protected against early motor and general neurological impairments observed in the open field and neurological score of severity, respectively, and late deficits in rotarod test. Melatonin prevented the NaDMDC-induced alterations in the striatal tyrosine hydroxylase immunocontent. Melatonin also protected against increased levels of oxidative stress markers (4-hydroxynonenal and 3-nitrotyrosine) in the striatum, as well as the NaDMDC-induced increase of 4-hydroxynonenal and TNF, markers of oxidative stress and inflammation, respectively, in the olfactory bulb. These results further detail the mechanisms underlying NaDMDC toxicity and demonstrate the neuroprotective effects of melatonin against the neuronal damage induced by NaDMDC., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glutathione (GSH) is a major cellular antioxidant molecule participating in several biological processes, including immune function. In this study, we investigated the importance of GSH to oysters Crassostrea gigas immune response. Oysters were treated with the GSH-synthesis inhibitor buthionine sulfoximine (BSO), and the function of immune cells and mortality were evaluated after a bacterial challenge with different Vibrio species. BSO caused a moderate decrease (20-40%) in GSH levels in the gills, digestive gland, and hemocytes. As expected, lower GSH decreased survival to peroxide exposure. Hemocyte function was preserved after BSO treatment, however, oysters became more susceptible to challenges with Vibrio anguillarum, V. alginolyticus, or V. harveyi, but not with V. parahaemolyticus and V. vulnificus, indicating a species-specific vulnerability. Our study indicates that in natural habitats or in mariculture farms, disturbances in GSH metabolism may pre-dispose oysters to bacterial infection, decreasing survival., 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 Ltd. All rights reserved.)

Methylglyoxal (MGO) is a highly reactive dicarbonyl molecule that promotes the formation of advanced glycation end products (AGEs), which are believed to play a key role in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. Here, Swiss mice were treated with MGO by intraperitoneal injection to investigate its effects on motor activity, mood, and cognition. Acute MGO treatment heavily decreased locomotor activity in the open field test at higher doses (80-200 mg/kg), an effect not observed at lower doses (10-50 mg/kg). Several alterations were observed 4 h after a single MGO injection (10-50 mg/kg): (a) plasma MGO levels were increased, (b) memory was impaired (object location task), (c) anxiolytic behavior was observed in the open field and marble burying test, and (d) depressive-like behavior was evidenced as evaluated by the tail suspension test. Biochemical alterations in the glutathione and glyoxalase systems were not observed 4 h after MGO treatment. Mice were also treated daily with MGO at 0, 10, 25 and 50 mg/kg for 11 days. From the 5th to the 11th day, several behavioral end points were evaluated, resulting in: (a) absence of motor impairment as evaluated in the open field, horizontal bars and pole test, (b) depressive-like behavior observed in the tail suspension test, and (c) cognitive impairments detected on working, short- and long-term memory when mice were tested in the Y-maze spontaneous alternation, object location and recognition tests, and step-down inhibitory avoidance task. An interesting finding was a marked decrease in dopamine levels in the prefrontal cortex of mice treated with 50 mg/kg MGO for 11 days, along with a ~ 25% decrease in the Glo1 content. The MGO-induced dopamine depletion in the prefrontal cortex may be related to the observed memory deficits and depressive-like behavior, an interesting topic to be further studied as a potentially novel route for MGO toxicity.

Memory impairment is a feature of several diseases and detrimental as aging population have increased worldwide. Sustained advanced glycation end products (AGEs) receptor (RAGE) activation triggers the production of reactive oxygen species and inflammatory response, leading to neuronal dysfunction and neurodegenerative disorders. Methylglyoxal (MGO) is the most relevant and reactive glycating agent in vivo, leading to the formation of AGEs. Here, we investigated the role of RAGE on the memory impairment induced by MGO. Swiss female mice were treated for 11 days with MGO, FPS‑ZM1 (a high‑affinity RAGE antagonist), or the combination of both. Locomotor activity was not impaired by the treatments, as evaluated by the open field and spontaneous alternation test. MGO treatment impaired short‑ and long‑term spatial memory in the object location task, caused deficits on the short‑term aversive memory in the step‑down inhibitory avoidance task, and decreased working memory performance as evaluated by the Y‑maze spontaneous alternation test. FPS‑ZM1 treatment abolished deficits on the short‑term aversive memory and working memory, but was unable to prevent the impairment in short‑term or long‑term spatial memory. Since the addition of RAGE antagonist in co‑treatment with MGO protected mice from the aversive and working memory deficits, AGEs generated by the MGO treatment would be involved in the memory impairment due to RAGE activation. Therefore, further studies are required to establish the involvement of RAGE in the MGO‑induced memory impairment. Nevertheless, our results suggested FPS‑ZM1 treatment as a promising new therapeutic strategy to prevent cognitive dysfunction caused by dicarbonyl stress, further investigation is required to confirm our findings.

Thioredoxin interacting protein (TXNIP) binds to thioredoxin thereby limiting its activity, but it also promotes internalization of glucose transporters, participates in inflammasome activation, and controls autophagy. Published data and this work demonstrate that TXNIP responds to a number of apparently unrelated stresses, such as serum deprivation, pH change, and oxidative, osmotic and carbonyl stress. Interestingly, we noticed that hyperosmotic (NaCl) and carbonyl (methylglyoxal, MGO) stresses in HT22 neuronal cells produced a rapid loss of TXNIP (half-life ∼12 min), prompting us to search for possible mechanisms controlling this TXNIP loss, including pH change, serum deprivation, calcium metabolism and inhibition of the proteasome and other proteases, autophagy and MAPKs. None of these routes stopped the TXNIP loss induced by hyperosmotic and carbonyl stress. Besides transcriptional, translational and microRNA regulation, there is evidence indicating that mTOR and AMPK also control TXNIP expression. Indeed, AMPK-deficient mouse embryonic fibroblasts failed to respond to phenformin (AMPK activator) and compound C (AMPK inhibitor), while rapamycin induced a marked increase in TXNIP levels, confirming the known AMPK/mTOR control over TXNIP. However, the TXNIP loss induced by NaCl or MGO were observed even in AMPK deficient MEFs or after mTOR inhibition, indicating AMPK/mTOR does not participate in this rapid TXNIP loss. These results suggest that rapid TXNIP loss is a general and immediate response to stress that can improve energy availability and antioxidant protection, eventually culminating in better cell survival., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Hyperosmotic stress is an important pathophysiologic condition in diabetes, severe trauma, dehydration, infection, and ischemia. Furthermore, brain neuronal cells face hyperosmotic stress in ageing and Alzheimer's disease. Despite the enormous importance of knowing the homeostatic mechanisms underlying the responses of nerve cells to hyperosmotic stress, this topic has been underrepresented in the literature. Recent evidence points to autophagy induction as a hallmark of hyperosmotic stress, which has been proposed to be controlled by mTOR inhibition as a consequence of AMPK activation. We previously showed that methylglyoxal induced a decrease in the antioxidant proteins thioredoxin 1 (Trx1) and glyoxalase 2 (Glo2), which was mediated by AMPK-dependent autophagy. Thus, we hypothesized that hyperosmotic stress would have the same effect., Methods: HT22 hippocampal nerve cells were treated with NaCl (37, 75, or 150 mM), and the activation of the AMPK/mTOR pathway was investigated, as well as the levels of Trx1 and Glo2. To determine if autophagy was involved, the inhibitors bafilomycin (Baf) and chloroquine (CQ), as well as ATG5 siRNA, were used. To test for AMPK involvement, AMPK-deficient mouse embryonic fibroblasts (MEFs) were used., Results: Hyperosmotic stress induced a clear increase in autophagy, which was demonstrated by a decrease in p62 and an increase in LC3 lipidation. AMPK phosphorylation, linked to a decrease in mTOR and S6 ribosomal protein phosphorylation, was also observed. Deletion of AMPK in MEFs did not prevent autophagy induction by hyperosmotic stress, as detected by decreased p62 and increased LC3 II, or mTOR inhibition, inferred by decreased phosphorylation of P70 S6 kinase and S6 ribosomal protein. These data indicating that AMPK was not involved in autophagy activation by hyperosmotic stress were supported by a decrease in p S555 -ULK1, an AMPK phosphorylation site. Trx1 and Glo2 levels were decreased at 6 and 18 h after treatment with 150 mM NaCl. However, this decrease in Trx1 and Glo2 in HT22 cells was not prevented by autophagy inhibition by Baf, CQ, or ATG5 siRNA. AMPK-deficient MEFs under hyperosmotic stress presented the same Trx1 and Glo2 decrease as wild-type cells., Conclusion: Hyperosmotic stress induced AMPK activation, but this was not responsible for its effects on mTOR activity or autophagy induction. Moreover, the decrease in Trx1 and Glo2 induced by hyperosmotic stress was independent of both autophagy and AMPK activation.

Oxidative stress and mitochondrial dysfunction are critical events in neurodegenerative diseases; therefore, molecules that increase cellular antioxidant defenses represent a future pharmacologic strategy to counteract such conditions. The aim of this study was to investigate the potential protective effect of (PhSe) 2 on mouse hippocampal cell line (HT22) exposed to tert-BuOOH (in vitro model of oxidative stress), as well as to elucidate potential mechanisms underlying this protection. Our results showed that tert-BuOOH caused time- and concentration-dependent cytotoxicity, which was preceded by increased oxidants production and mitochondrial dysfunction. (PhSe) 2 pre-incubation significantly prevented these cytotoxic events and the observed protective effects were paralleled by the upregulation of the cellular glutathione-dependent antioxidant system: (PhSe) 2 increased GSH levels (> 60%), GPx activity (6.9-fold) and the mRNA expression of antioxidant enzymes Gpx1 (3.9-fold) and Gclc (2.3-fold). Of note, the cytoprotective effect of (PhSe) 2 was significantly decreased when cells were treated with mercaptosuccinic acid, an inhibitor of GPx, indicating the involvement of GPx modulation in the observed protective effect. In summary, the present findings bring out a new action mechanism concerning the antioxidant properties of (PhSe) 2 . The observed upregulation of the glutathione-dependent antioxidant system represents a future pharmacologic possibility that goes beyond the well-known thiol-peroxidase activity of this compound., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, we evaluated the effects of high-intensity swimming in an experimental model of acute lung injury (ALI) induced by lipopolysaccharide (LPS) on lung inflammation and antioxidant defenses. Balb/C male mice were submitted to exercise (30 min/day, 5 days/week, for a period of 3 weeks) prior to LPS instillation in the lung. Twenty-four hours after delivery of LPS (10 μg/animal), mice were euthanized and bronchoalveolar fluid (BALF) was obtained for cell counting and analysis of cytokines by ELISA. Lung tissue was used to evaluate antioxidant defenses. LPS instillation resulted in an increase in total and mononuclear cells, IL-1β, TNF-α, and IL-6 in BALF. LPS instillation also altered IL-10 and IL-ra levels in BALF and induced antioxidant defenses (glutathione, superoxide dismutase, catalase, and glutathione peroxidase) in the lung. Protein carbonyl increased in the LPS-treated animals. High-intensity swimming prevented all these changes induced by LPS. Significance: Therefore, this experimental protocol of high-intensity swimming showed a protective effect on ALI, decreasing inflammatory processes and preventing disturbances in antioxidant defenses into the lungs.

Systemic inflammation triggered by lipopolysaccharide (LPS) administration disrupts blood-brain barrier (BBB) homeostasis in animal models. This event leads to increased susceptibility of several encephalic structures to potential neurotoxicants present in the bloodstream. In this study, we investigated the effects of alternate intraperitoneal injections of LPS on BBB permeability, social recognition memory and biochemical parameters in the striatum 24 h and 60 days after treatments. In addition, we investigated whether the exposure to a moderate neurotoxic dose of the herbicide paraquat could potentiate LPS-induced neurotoxicity. LPS administration caused a transient disruption of BBB integrity, evidenced by increased levels of exogenously administered sodium fluorescein in the striatum. Also, LPS exposure caused delayed impairment in social recognition memory (evaluated at day 38 after treatments) and increase in the striatal levels of 3-nitrotyrosine. These events were observed in the absence of significant changes in motor coordination and in the levels of tyrosine hydroxylase (TH) in the striatum and substantia nigra. PQ exposure, which caused a long-lasting decrease of striatal mitochondrial complex I activity, did not modify LPS-induced behavioral and striatal biochemical changes. The results indicate that systemic administration of LPS causes delayed social recognition memory deficit and striatal nitrosative stress in adult mice and that the coexposure to a moderately toxic dose of PQ did not magnify these events. In addition, PQ-induced inhibition of striatal mitochondrial complex I was also not magnified by LPS exposure, indicating the absence of synergic neurotoxic effects of LPS and PQ in this experimental model.

Traumatic brain injury (TBI) is a worldwide core public health problem affecting mostly young male subjects. An alarming increase in incidence has turned TBI into a leading cause of morbidity and mortality in young adults as well as a tremendous resource burden on the health and welfare sector. Hormone dysfunction is highly prevalent during the acute phase of severe TBI. In particular, investigation of the luteinizing hormone (LH) and testosterone levels during the acute phase of severe TBI in male has identified a high incidence of low testosterone levels in male patients (36.5-100%) but the prognostic significance of which remains controversial. Two independent studies showed that normal or elevated levels of LH levels earlier during hospitalization are significantly associated with higher mortality/morbidity. The association between LH levels and prognosis was independent of other predictive variables such as neuroimaging, admission Glasgow coma scale, and pupillary reaction. The possible mechanisms underlying this association and further research directions in this field are discussed. Overall, current data suggest that LH levels during the acute phase of TBI might contribute to accurate prognostication and further prospective multicentric studies are required to develop more sophisticated predictive models incorporating biomarkers such as LH in the quest for accurate outcome prediction following TBI. Moreover, the potential therapeutic benefits of modulating LH during the acute phase of TBI warrant investigation.

Thioredoxin (Trx) and glyoxalase (Glo) systems have been suggested to be molecular targets of methylglyoxal (MGO). This highly reactive endogenous compound has been associated with the development of neurodegenerative pathologies and cell death. In the present study, the glutathione (GSH), Trx, and Glo systems were investigated to understand early events (0.5-3 h) that may determine cell fate. It is shown for the first time that MGO treatment induces an increase in glutathione reductase (GR) protein in hippocampal slices (1 h) and HT22 nerve cells (0.5 and 2.5 h). Thioredoxin interacting protein (Txnip), thioredoxin reductase (TrxR), Glo1, and Glo2 were markedly increased (2- to 4-fold) in hippocampal slices and 1.2- to 1.3-fold in HT22 cells. This increase in protein levels in hippocampal slices was followed by a corresponding increase in GR, TrxR, and Glo1 activities, but not in HT22 cells. In these cells, GR and TrxR activities were decreased by MGO. This result is in agreement with the idea that MGO can affect the Trx/TrxR reducing system, and now we show that GR and Txnip can also be affected by MGO. Impairment in the GR or TrxR reducing capacity can impair peroxide removal by glutathione peroxidase and peroxiredoxin, as both peroxidases depend on reduced GSH and Trx, respectively. In this regard, inhibition of GR and TrxR by 2-AAPA or auranofin, respectively, potentiated MGO toxicity in differentiated SH-SY5Y cells. Overall, MGO not only triggers a clear defense response in hippocampal slices and HT22 cells but also impairs the Trx/TrxR and GSH/GR reducing couples in HT22 cells. The increased MGO toxicity caused by inhibition of GR and TrxR with specific inhibitors, or their inhibition by MGO treatment, supports the notion that both reducing systems are relevant molecular targets of MGO.

Nrf2 is a well-known transcription factor controlling a number of antioxidant defense-related genes, which is understudied in bivalves. In this study, oysters Crassostrea gigas were exposed for 24, 48 and 96 h to 10 or 30 μM tert-butylhydroquinone (tBHQ), a classic Nrf2 activator. At 96 h, a clear induction of GSH-related antioxidant defenses was observed in gills of tBHQ-exposed animals, including GSH, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR). Unexpectedly, the activities of GST, GPx and GR were significantly decreased 24 h after tBHQ treatment, suggesting a possible inhibition, which was supported by in vitro experiments. GR mRNA (24 h) and protein levels (24 and 96 h) were increased by tBHQ treatment, confirming its induction, possibly by the Nrf2 pathway. The conserved domains at C. gigas Keap1 and Nrf2 proteins and the clear induction of GSH-related antioxidant defenses by tBHQ, a classical Nrf2 inducer, support the idea of a functional Nrf2/Keap1 pathway in bivalves. tBHQ also proved to be a tool to explore redox regulatory mechanisms in bivalves., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thiol homeostasis has a critical role in the maintenance of proper cellular functions and survival, being coordinated by the action of several reductive enzymes, including glutathione (GSH)/glutathione reductase (GR) and thioredoxin (Trx)/thioredoxin reductase (TrxR) systems. Here, we investigated the effects of the GR inhibitor 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) on the activity of thiol reductases (GR and TrxR), redox balance and mitochondrial function of A172 glioblastoma cells. 2-AAPA inhibited cell GR (IC 50 =6.7μM) and TrxR (IC 50 =8.7μM). A significant decrease in the cellular ability to decompose cumene hydroperoxide was observed and associated to a greater susceptibility to this peroxide. The redox state of peroxiredoxins (Prx1, Prx2 and Prx3) was markedly shifted to dimer 30min after treatment with 100μM 2-AAPA, an event preceding 2-AAPA-induced decrease in cell viability. Furthermore, mitochondrial function was also severely impaired, leading to a decrease in the respiratory control ratio, reserve capacity, and ATP synthesis-coupled respiration, as well as an increase in mitochondrial membrane potential. Our results indicate that inhibition of GR and TrxR activities, disruption of the ability to detoxify peroxides, increased oxidation of Prxs, as well as compromised mitochondrial function represent early events mediating 2-AAPA toxicity to A172 glioblastoma cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. We previously showed that MGO treatment targets the thioredoxin and the glyoxalase systems, leading to a decrease in Trx1 and Glo2 proteins in immortalized mouse hippocampal HT22 nerve cells. Here, we propose that autophagy is the underlying mechanism leading to Glo2 and Trx1 loss induced by MGO. The autophagic markers p62, and the lipidated and active form of LC3, were increased by MGO (0.5mM). Autophagy inhibition with bafilomycin or chloroquine prevented the decrease in Trx1 and Glo2 at 6 and 18h after MGO treatment. Proteasome inhibition by MG132 exacerbated the effect of MGO on Trx1 and Glo2 degradation (18h), further suggesting a role for autophagy. ATG5 small interfering RNA protected Trx1 and Glo2 from MGO-induced degradation, confirming Trx1 and Glo2 loss is mediated by autophagy. In the search for the signals that control autophagy, we found that AMPK activation, a known autophagy inducer, was markedly increased by MGO treatment. AMPK activation was confirmed by increased acetyl coenzyme A carboxylase phosphorylation, a direct AMPK substrate and by decreased mTOR phosphorylation, an indirect marker of AMPK activation. To confirm that MGO-mediated Trx1 and Glo2 degradation was AMPK-dependent, AMPK-deficient mouse embryonic fibroblasts (MEFs) were treated with MGO. Wildtype MEFs presented the expected decrease in Trx1 and Glo2, while MGO was ineffective in decreasing these proteins in AMPK-deficient cells. Overall, the data indicate that MGO activates autophagy in an AMPK-dependent manner, and that autophagy was responsible for Trx1 and Glo2 degradation, confirming that Trx1 and Glo2 are molecular targets of MGO., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Analysis of the Pacific oyster Crassostrea gigas annotated genome revealed genes with conserved sequences belonging to typical cap 'n' collar Nrf2 domain, a major player in antioxidant protection, and domains belonging to Nrf2 cytoplasmic repressor (Keap1), but little is known about Nrf2/Keap1 induction in bivalves. C. gigas were exposed to waterborne 10 and 30μM curcumin, a known inducer of the mammalian Nrf2. Curcumin disappeared from the seawater after 10h, and accumulated in the gills (10h) and digestive gland (10-96h). A clear induction of glutathione (GSH)-related antioxidant defenses was observed at 96h in the gills of curcumin exposed animals (10 and 30μM), including GSH levels, and the activity of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). This response was completely absent in the digestive gland, in line with the idea that bivalve gills act as a major site for antioxidant protection under acute exposure. The relative mRNA levels coding glutamate-cysteine ligase, GR, GPx2 and GSTpi were clearly induced by curcumin treatment (30μM, 24h). Curcumin pre-treatment for 96h increased oyster resistance to cumene hydroperoxide, but neither Nrf2 nor Keap1 genes were modulated by curcumin. However, the conserved sequences belonging to typical Nrf2 and Keap1 domains, and the notorious induction of antioxidant defense-related genes known to be controlled by Nrf2 in mammals, indicates a functional Nrf2/Keap1 pathway in bivalves, and curcumin seems to be a new tool to investigate the antioxidant response in bivalves., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study investigated the effects of hypoxia on oxidative stress response and immune function in mussels Perna perna exposed to air for 6, 12, 24 and 48 h. In air-exposed mussels, the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione reductase (GR) activities were lower in gill tissues (24-48 h) and digestive gland (12 h), while the glutathione peroxidase and GR activities were increased in the digestive gland (48 h). In both tissues, aerial exposure promoted a rapid (6 h) and persistent (up to 48 h) increase of glutathione levels. Decreased hemocyte count and viability, as well as increased phagocytic activity and cellular adhesion capacity were detected after prolonged aerial exposure (>12 h). In summary, induction of thiol pools, altered antioxidant enzyme activities, and activation of immune responses were detected in hypoxia exposed brown mussels, indicating hypoxia induced tissue-specific responses in both antioxidant and immune systems., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pramipexole (PPX), a dopamine D2/3 receptor preferring agonist, is currently in use for the treatment of Parkinson's disease symptoms and restless legs syndrome. Recently, anti-inflammatory properties of PPX have been shown in an autoimmune model of multiple sclerosis, and case reports indicate PPX ameliorates depressive symptoms. Since peripheral inflammation is known to induce depression-like behavior in rodents, we assessed the potential antidepressant effect of PPX in an inflammatory model of depression induced by LPS. Repeated (daily for 7days, 1mg/kg, i.p.), but not acute (1h before LPS) treatment with PPX abolished the depression-like behavior induced by LPS (0.1mg/kg, i.p.) in the forced swim test, and the anhedonic behavior in the splash test. Interestingly, PPX per se decreased interleukin 1β levels and reversed LPS-induced increase in its content in mice hippocampus⋅ Repeated PPX treatment also prevented the increase in hippocampal levels of the 3-nitrotyrosine protein adducts induced by LPS. Haloperidol (0.2mg/kg, i.p.) and sulpiride (50mg/kg, i.p.) were unable to prevent the antidepressant-like effect of PPX in LPS-treated mice. Altogether, these results suggest that the observed antidepressant-like effect of PPX in LPS-treated mice may be dependent on its anti-inflammatory properties and may not be related to dopamine D2 receptor activation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective Given that the literature data indicates that ascorbic acid may have an anxiolytic effect, we hypothesized that a single oral administration of ascorbic acid could acutely affect emotional states. Methods The effects of acid ascorbic supplementation on anxiety and other emotional states were evaluated by the State‑Trait Anxiety Inventory (STAI), and Visual Analogue Mood Scale (VAMS). Immediately before, and 2 hours after receiving a single ascorbic acid dose (1000 mg) or placebo, 142 graduate students were evaluated by the STAI and VAMS in a randomized, double‑blind, placebo‑controlled trial. Results No changes from basal levels were observed in the STAI state‑anxiety or VAMS scores. However, the ingestion of ascorbic acid by the 25% more anxious healthy subjects (women; 14 control and 23 ascorbic acid), as defined by the STAI trait‑anxiety scale, produced a significant reduction from baseline anxiety scores in the STAI state‑anxiety scale and VAMS anxiety subscale. The study evaluated a small sample with narrow sociodemographic characteristics, composed mainly of healthy young females (> 94%) enrolled in post‑graduation courses, without controlling diet, physical activity, and formal psychiatric diagnosis., Conclusions: Despite the sample size limitation, this study provides the first evidence of an acute anxiolytic effect of ascorbic acid. Broader population studies are required to evaluate the clinical relevance of presented data.

α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound.

Metal contamination at low levels is an important issue because it usually produces health and environmental effects, either positive or deleterious. Contamination of surface waters with copper (Cu) is a worldwide event, usually originated by mining, agricultural, industrial, commercial, and residential activities. Water quality criteria for Cu are variable among countries but allowed limits are generally in the μg/L range, which can disrupt several functions in the early life-stages of fish species. Behavioral and biochemical alterations after Cu exposure have also been described at concentrations close to the allowed limits. Aiming to search for the effects of Cu in the range of the allowed limits, larvae and adult zebrafish (Danio rerio) were exposed to different concentrations of dissolved Cu (nominally: 0, 5, 9, 20 and 60μg/L; measured: 0.4, 5.7, 7.2 16.6 and 42.3μg/L, respectively) for 96h. Larvae swimming and body length, and adult behavior and biochemical biomarkers (activity of glutathione-related enzymes in gills, muscle, and brain) were assessed after Cu exposure. Several effects were observed in fish exposed to 9μg/L nominal Cu, including increased larvae swimming distance and velocity, abolishment of adult inhibitory avoidance memory, and decreased glutathione S-transferase (GST) activity in gills of adult fish. At the highest Cu concentration tested (nominally: 60μg/L), body length of larvae, spatial memory of adults, and gill GST activity were decreased. Social behavior (aggressiveness and conspecific interaction), and glutathione reductase (GR) activity were not affected in adult zebrafish. Exposure to Cu, at concentrations close to the water quality criteria for this metal in fresh water, was able to alter larvae swimming performance and to induce detrimental effects on the behavior of adult zebrafish, thus indicating the need for further studies to reevaluate the currently allowed limits for Cu in fresh water., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bivalves are animals with worldwide distribution. Although they play key roles in economic activities, human feeding and environmental studies, there is a considerable lack of knowledge about the relationship between their immune system and antioxidant defenses. Here, we performed an in vitro experiment where Crassostrea gigas hemocytes were exposed to the electrophilic compound 1-chloro-2,4-dinitrobenzene (CDNB, 0.1-50 μM) for one hour. CDNB treatment clearly disturbed thiol homeostasis, causing a concentration-dependent decrease in the glutathione (GSH) content and a decrease in the activity of two thiol reductases, glutathione reductase (GR - 2.5 and 50 μM CDNB) and thioredoxin reductase (TrxR - only 50 μM CDNB). The MTT reduction assay showed that none of the CDNB concentrations tested significantly altered cell viability. However, there was a decrease in the hemocyte's ability to uptake the neutral red dye, which indicates lysosomal impairment (≥12.5 μM CDNB). Cellular immunocompetence was further investigated and, despite the lower GSH content, GR activity and impairment in lysosome integrity, hemocyte functions (adhesion capacity, phagocytosis of latex beads and laminarin-induced ROS production) were preserved in the 2.5 and 12.5 μM CDNB treatments. These results suggest a minor importance of thiol pools and GR activity in C. gigas hemocyte's immunocompetence, in an in vitro acute exposure model. The 50 μM CDNB treatment, however, significantly compromised all the measured hemocyte functions. This response was associated with TrxR inhibition, increased lysosome impairment, decreased GSH content, and lower GR activity. Therefore, it seems that TrxR may be particularly important for the hemocyte function, or, alternatively, it is only affected when a deeply aggravated scenario in thiol homeostasis is set up. Such findings point out the need for further studies towards a better understanding of antioxidant and immune defenses interactions in bivalve cellular systems., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In Brazil B5 blend (5% biodiesel and 95% diesel oil) has been adopted as mandatory fuel since 2010 for automotive vehicles. Since little is known about the effects of B5 exposure can promote on antioxidant system of marine biota this study aimed to assess if B5 can generate modifications in antioxidant parameters of mussels Perna perna. To address this question mussels were exposed to two concentrations of B5 (0.01 mL L(-1) and 0.1 mL L(-1)) for 6h, 12h, 48 h and 168 h. Then the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were evaluated in gills and digestive gland as well as the contents of glutathione (GSH) and lipid peroxidation by measuring the malondialdehyde concentration (MDA). In the gills, GST activity decreased after 48 h and GR after 12h of exposure to B5. In digestive glands, the activities of SOD, GPx and GR were changed due to treatments. GSH concentration increased in digestive gland after 6h and 12h and in gills after 48 h for B5 0.1 mL L(-1) and after 168 h in the digestive gland for B5 0.01 mL L(-1) treatment. No lipid peroxidation was detected. The integrated biomarker response index (IBR) evidenced a B5 effect in the digestive gland after 168 h of exposure. Regarding the experimental conditions and species used in this study, long-term exposure to B5 is apparently more likely to affect the parameters tested in P. perna mussels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Disperse Red 1 (DR1) is an azo dye that can reach the aquatic environment through the discharge of textile industrial wastewaters. It has been tested in Daphnia similis and shown to be highly toxic. Cytochrome P450 (CYP) is a class of enzymes involved in phase I of detoxification, while glutathione S-transferase (GST) are a class of phase II enzymes. No information about phase I or II dye metabolism in microcrustacea were found in the literature. In this study we identified CYP and GST enzymes involved in the metabolism of DR1 in juveniles of D. similis. Using spectrophotometric analysis we showed that 50 % of the dye was absorbed by the organisms, which could be confirmed by the reddish color of animals exposed to DR1, however adsorption cannot be ruled out. GST activity increased from 280 to 615 nmol(-1 )min(-1 )mg when D. similis were exposed for 48 h to 0.2 mg L(-1) DR1 and from 274 to 815 nmol(-1) min(-1 )mg when exposed to 5 mg L(-1). Data clearly demonstrate that exposure to DR1 can stimulate a strong induction of GST activity, whose participation in DR1 metabolism needs to be confirmed. The induction of GST activity seems to be dependent on CYP activity, since treatment with SKF535A, a CYP inhibitor, blocked the DR1-dependent GST induction. We speculate that GST is involved in DR1 metabolism in Daphnia and that CYP activity is necessary to induce GST-activity, which is an indirect evidence of its role in the biotransformation of DR1.

Organic peroxide elimination in eukaryotes essentially depends on glutathione peroxidase (GPx) and peroxiredoxin (Prx) enzymes, which are supported by their respective electron donors, glutathione (GSH) and thioredoxin (Trx). This system depends on the ancillary enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) to maintain GSH and Trx in their reduced state. This study discusses the biological importance of GR and TrxR in supporting GPx and Prx during cumene hydroperoxide (CHP) exposure in brown mussel Perna perna. ZnCl2 or 1-chloro-2,4-dinitrobenze (CDNB) was used to decrease GR and TrxR activities in gills, as already reported with mammals and bivalves. ZnCl2 exposure lowered GR activity (28%), impaired the in vivo CHP decomposition and decreased the survival rates under CHP exposure. CDNB decreased GR (54%) and TrxR (73%) activities and induced glutathione depletion (99%), promoting diminished peroxide elimination and survival rates at a greater extent than ZnCl2. CDNB also increased the susceptibility of hemocytes to CHP toxicity. Despite being toxic and causing mortality at longer exposures, short (2 h) exposure to CHP promoted an up regulation of GSH (50 and 100 μM CHP) and protein-thiol (100 μM CHP) levels, which was blocked by ZnCl2 or CDNB pre-exposure. Results highlight the biological importance of GSH, GR and TrxR in supporting GPx and Prx activities, contributing to organic peroxides elimination and mussel survival under oxidative challenges. To our knowledge, this is the first work that demonstrates, albeit indirectly, the biological importance of GPx/GR/GSH and Prx/TrxR/Trx systems on in vivo organic peroxide elimination in bivalves., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Manganese (Mn) exposure is related to industrial activities, where absorption by inhalation has high relevance. Manganism, a syndrome caused as a result of excessive accumulation of Mn in the central nervous system, has numerous symptoms similar to those seen in idiopathic Parkinson disease (IPD). Some of these symptoms, such as learning, memory, sensorial, and neurochemical changes, appear before the onset of motor deficits in both manganism and IPD. The aim of this study was to evaluate the possible neuroprotective effects of curcumin against behavioral deficits induced by Mn toxicity in young (2 months old) Swiss mice. We evaluated the effect of chronic inhalation of a Mn mixture [Mn(OAc)3 and MnCl2 (20:40 mM)], 1 h/session, three times a week, over a 14-week period on behavioral and neurochemical parameters. Curcumin was supplemented in the diet (500 or 1,500 ppm in food pellets). The Mn disrupted the motor performance evaluated in the single-pellet reach task, as well as the short- and long-term spatial memory evaluated in the step-down inhibitory avoidance task. Surprisingly, curcumin also produced similar deleterious effects in such behavioral tests. Moreover, the association of Mn plus curcumin significantly increased the levels of Mn and iron, and decreased the levels of dopamine and serotonin in the hippocampus. These alterations were not observed in the striatum. In conclusion, the current Mn treatment protocol resulted in mild deficits in motor and memory functions, resembling the early phases of IPD. Additionally, curcumin showed no beneficial effects against Mn-induced disruption of hippocampal metal and neurotransmitter homeostasis.

Zinc demonstrates protective and antioxidant properties at physiological levels, although these characteristics are not attributed at moderate or high concentrations. Zinc toxicity has been related to a number of factors, including interference with antioxidant defenses. In particular, the inhibition of glutathione reductase (GR) has been suggested as a possible mechanism for acute zinc toxicity in bivalves. The present work investigates the biochemical effects of a non-lethal zinc concentration on antioxidant-related parameters in gills of brown mussels Perna perna exposed for 21 days to 2.6 μM zinc chloride. After 2 days of exposure, zinc caused impairment of the antioxidant system, decreasing GR activity and glutathione levels. An increase in antioxidant defenses became evident at 7 and 21 days of exposure, as an increase in superoxide dismutase and glutathione peroxidase activity along with restoration of glutathione levels and GR activity. After 7 and 21 days, an increase in cellular peroxides and lipid peroxidation end products were also detected, which are indicative of oxidative damage. Changes in GR activity contrasts with protein immunoblotting data, suggesting that zinc produces a long lasting inhibition of GR. Contrary to the general trend in antioxidants, levels of peroxiredoxin 6 decreased after 21 days of exposure. The data presented here support the hypothesis that zinc can impair thiol homeostasis, causes an increase in lipid peroxidation and inhibits GR, imposing a pro-oxidant status, which seems to trigger homeostatic mechanisms leading to a subsequent increase on antioxidant-related defenses., (© 2013.)

Traumatic brain injury (TBI) is frequently associated with abnormal blood-brain barrier function, resulting in the release of factors that can be used as molecular biomarkers of TBI, among them GFAP, UCH-L1, S100B, and NSE. Although many experimental studies have been conducted, clinical consolidation of these biomarkers is still needed to increase the predictive power and reduce the poor outcome of TBI. Interestingly, several of these TBI biomarkers are oxidatively modified to carbonyl groups, indicating that markers of oxidative stress could be of predictive value for the selection of therapeutic strategies. Some drugs such as corticosteroids and progesterone have already been investigated in TBI neuroprotection but failed to demonstrate clinical applicability in advanced phases of the studies. Dietary antioxidants, such as curcumin, resveratrol, and sulforaphane, have been shown to attenuate TBI-induced damage in preclinical studies. These dietary antioxidants can increase antioxidant defenses via transcriptional activation of NRF2 and are also known as carbonyl scavengers, two potential mechanisms for neuroprotection. This paper reviews the relevance of redox biology in TBI, highlighting perspectives for future studies.

This work evaluates the effects of caging, a known confinement stress, in Nile tilapia (Oreochromis niloticus) during an environmental study in Cubatão river, southern Brazil. Caging animals for 7 days, regardless of being at the reference or at a contaminated site, resulted in lower levels of antioxidant-related defenses (glutathione, glutathione S-transferase, glucose-6-phosphate dehydrogenase) in liver and physiological parameters (blood glucose and lactate) as compared with free-swimming animals. Higher hepatic glutathione reductase activity and elevated Hb content could be associated to contaminant exposure. In conclusion, the confinement stress in caged Nile tilapia biochemical and physiological disturbances, acting as a confounding factor in field studies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Glutathione (GSH) displays a broad range of functions, among them a role as a neuromodulator with some neuroprotective properties. Taking into account that oxidative stress has been associated with depressive disorders, this study investigated the possibility that GSH, a major cell antioxidant, elicits an antidepressant-like effect in mice. Thus, GSH was administered by i.c.v. route to mice that were tested in the forced swimming test and in the tail suspension test, two predictive tests for antidepressant drug activity. In addition, GSH metabolism and the redox environment were modulated in order to study the possible mechanisms underlying the effects of GSH in the forced swimming test. The administration of GSH decreased the immobility time in the forced swimming test (300-3000nmol/site) and tail suspension test (100-1000nmol/site), consistent with an antidepressant-like effect. GSH depletion elicited by l-buthionine sulfoximine (3.2μmol/site, i.c.v.) did not alter the antidepressant-like effect of GSH, whereas the inhibition of extracellular GSH catabolism by acivicin (100nmol/site, i.c.v.) prevented the antidepressant-like effect of GSH. Moreover, a sub-effective dose (0.01nmol/site, i.c.v.) of the oxidizing agent DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) potentiated the effect of GSH (100nmol/site, i.c.v.), while the pretreatment (25-100mg/kg, i.p.) with the reducing agent DTT (dl-dithiothreitol) prevented the antidepressant-like effect of GSH (300nmol/site, i.c.v.). DTNB (0.1nmol/site, i.c.v.), produced an antidepressant-like effect, per se, which was abolished by DTT (25mg/kg, i.p.). The results show, for the first time, that centrally administered GSH produces an antidepressant-like effect in mice, which can be modulated by the GSH metabolism and the thiol/disulfide reagents. The redox environment may constitute a new venue for future antidepressant-drug development., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes.

Disturbances in antioxidant defenses decrease cellular protection against oxidative stress and jeopardize cellular homeostasis. To knock down the antioxidant defenses of Pacific oyster Crassostrea gigas, animals were pre-treated with 1-chloro-2,4-dinitrobenzene (CDNB) and further challenged with pro-oxidant menadione (MEN). CDNB pre-treatment (10 μM for 18 h) was able to consume cellular thiols in gills, decreasing GSH (53%) and decrease protein thiols (25%). CDNB pre-treatment also disrupted glutathione reductase and thioredoxin reductase activity in the gills, but likewise strongly induced glutathione S-transferase activity (270% increase). Surprisingly, hemocyte viability was greatly affected 24 h after CDNB removal, indicating a possible vulnerability of the oyster immune system to electrophilic attack. New in vivo approaches were established, allowing the identification of higher rates of GSH-CDNB conjugate export to the seawater and enabling the measurement of the organic peroxide consumption rate. CDNB-induced impairment in antioxidant defenses decreased the peroxide removal rate from seawater. After showing that CDNB decreased gill antioxidant defenses and increased DNA damage in hemocytes, oysters were further challenged with 1 mM MEN over 24 h. MEN treatment did not affect thiol homeostasis in gills, while CDNB pre-treated animals recovered GSH and PSH to the control level after 24 h of depuration. Interestingly, MEN intensified GSH and PSH loss and mortality in CDNB-pre-treated animals, showing a clear synergistic effect. The superoxide-generating one-electron reduction of MEN was predominant in gills and may have contributed to MEN toxicity. These results support the idea that antioxidant-depleted animals are more susceptible to oxidative attack, which can compromise survival. Data also corroborate the idea that gills are an important detoxifying organ, able to dispose of organic peroxides, induce phase II enzymes, and efficiently export GSH-CDNB conjugates., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cells are endowed with several overlapping peroxide-degrading systems whose relative importance is a matter of debate. In this study, three different sources of neural cells (rat hippocampal slices, rat C6 glioma cells, and mouse N2a neuroblastoma cells) were used as models to understand the relative contributions of individual peroxide-degrading systems. After a pretreatment (30 min) with specific inhibitors, each system was challenged with either H₂O₂ or cumene hydroperoxide (CuOOH), both at 100 μM. Hippocampal slices, C6 cells, and N2a cells showed a decrease in the H₂O₂ decomposition rate (23-28%) by a pretreatment with the catalase inhibitor aminotriazole. The inhibition of glutathione reductase (GR) by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) significantly decreased H₂O₂ and CuOOH decomposition rates (31-77%). Inhibition of catalase was not as effective as BCNU at decreasing cell viability (MTT assay) and cell permeability or at increasing DNA damage (comet test). Impairing the thioredoxin (Trx)-dependent peroxiredoxin (Prx) recycling by thioredoxin reductase (TrxR) inhibition with auranofin neither potentiated peroxide toxicity nor decreased the peroxide-decomposition rate. The results indicate that neural peroxidatic systems depending on Trx/TrxR for recycling are not as important as those depending on GSH/GR. Dimer formation, which leads to Prx2 inactivation, was observed in hippocampal slices and N2a cells treated with H₂O₂, but not in C6 cells. However, Prx-SO₃ formation, another form of Prx inactivation, was observed in all neural cell types tested, indicating that redox-mediated signaling pathways can be modulated in neural cells. These differences in Prx2 dimerization suggest specific redox regulation mechanisms in glia-derived (C6) compared to neuron-derived (N2a) cells and hippocampal slices., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Selenium and copper are naturally occurring elements in the environment that have important roles in cellular function. Selenium is known for its role in antioxidant defense, whereas copper is a redox-active metal capable of acting as a pro-oxidant. We investigated the effects of short term selenium (Na(2)SeO(3)) supplementation (4 μg/L for 3 days) on antioxidant parameters of the blue mussel, Mytilus edulis, and its possible protective effects against a subsequent copper (CuSO(4)) exposure (56 μg/L for 3 days). Selenium supplementation caused a 4-fold increase in glutathione levels in gills. The activity of selenium-dependent glutathione peroxidase was modulated by selenium in gills (2-fold increase) and also in cell-free haemolymph (40% increase). Copper exposure produced decreases in protein thiol levels (35%) and in thioredoxin reductase activity (60%) in gills and induced an increase in DNA damage in haemocytes (70% increase in % tail DNA observed using the comet assay). The decrease in thioredoxin reductase activity may constitute a mechanism of copper toxicity in bivalves, warranting further investigation. Pre-treatment with selenium largely prevented these deleterious effects of copper on protein thiols, thioredoxin reductase activity and DNA damage. The results suggest that induction of key antioxidant defenses such as glutathione and selenium-dependent glutathione peroxidase, as a result of selenium supplementation, may play an important role in protection of aquatic organisms against oxidative stress., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Gender is one of the most important factors in mammalian development and response to exogenous agents. Although there is increasing evidence that health effects of toxic xenobiotics differ in prevalence or are manifested differently in male and female, the molecular mechanisms related to these events remain unclear. In order to investigate the possible influence of gender, male and female Wistar rats from the same litter were exposed to zinc chloride (5 mg/kg, i.p.) or malathion (250 mg/kg, i.p.) 24 hr prior to the analyses of biochemical parameters related to the cholinergic and glutathione-antioxidant systems in cerebral cortex and hippocampus. After treatments, acetylcholinesterase (AChE) activity was reduced in the hippocampus and cerebral cortex of male and female rats treated with malathion, but the effect was more pronounced in the male group. Glutathione reductase (GR) and γ-glutamyl-transpeptidase activities were reduced in the hippocampus of males and females and a gender-specific effect of malathion was seen for glutathione S-transferase (GST), which was decreased only in male hippocampus and cortex, and for male cortical GR. Zinc chloride treatment decreased AChE activity in male and female cerebral cortex, with no obvious effect in the hippocampus. Male-specific antioxidant-related enzyme activity reductions were seen after zinc treatment for cortical GR, GST, glutathione peroxidase and glucose-6-phosphate dehydrogenase; and hippocampal GR. These results clearly demonstrate a greater detrimental effect on antioxidant-related enzyme activities in male hippocampus and cerebral cortex when rats were acutely exposed to malathion and zinc, demonstrating that the research on gender-related differences in health effects caused by xenobiotic and/or essential elements requires further attention., (© 2010 The Authors. Basic & Clinical Pharmacology & Toxicology © 2010 Nordic Pharmacological Society.)

Malathion is an organophosphate (OP) pesticide whose toxicity depends on its bioactivation to malaoxon. Human malathion poisoning has been treated with oximes (mainly pralidoxime) in an attempt to reactivate OP-inhibited acetylcholinesterase (AChE). However, pralidoxime has shown unsatisfactory therapeutic effects in malathion poisoning and its routine use has been questioned. In this study, we evaluated the in vitro potency of standards and newly developed oximes in reactivating malaoxon-inhibited AChE derived from mouse brain supernatants. Malaoxon displayed a concentration-dependent inhibitory effect on mouse brain AChE (IC(50) = 2.36 microM), and pralidoxime caused a modest reactivating effect (30% of reactivation at 600 microM). Obidoxime and trimedoxime, as well as K047 and K075, displayed higher reactivating effects (from 55% to 70% of reactivation at 600 muM) when compared with pralidoxime. The results show that obidoxime, trimedoxime, K074 and K075 present higher reactivating effects on malaoxon-inhibited AChE under in vitro conditions when compared with pralidoxime. Taking into account the unsatisfactory effects of pralidoxime as antidotal treatment in malathion poisonings, the present results suggest that obidoxime, trimedoxime, K074 and K075 might be interesting therapeutic strategies to reactivate malaoxon-inhibited AChE in malathion poisonings.

There is increasing evidence that health effects of toxic metals, including methylmercury (MeHg), differ in prevalence or are manifested differently in men and women. The present study was aimed at investigating the potential differential susceptibility of male and female Swiss mice against MeHg-induced neurotoxicity, which was evaluated by biochemical (cerebellar oxidative stress-related parameters) and behavioral (locomotor activity and motor performance) variables. We also aimed to evaluate the potential protective effects of 17β-estradiol against such toxicity in MeHg-exposed male animals. MeHg exposure (40mg/L, diluted in tap water, during 2 weeks) decreased locomotor activity and motor performance in both male and female animals, but such phenomena were higher in males. 17β-estradiol co-treatment (10μg/animal, in alternate days) prevented MeHg-induced locomotor deficits in males. MeHg exposure caused a significant increase (60%) in cerebellar lipid peroxidation in male mice, but did not in females. In close agreement, MeHg exposure decreased (43%) cerebellar glutathione peroxidase activity in males, but did not in females. These events were prevented by 17β-estradiol administration. Cerebellar GR activity was increased (25%) in MeHg-exposed males and such event was partially prevented by 17β-estradiol administration. These results indicate that the low susceptibility of female mice to the neurotoxicity elicited by MeHg is linked to neuroprotective effects of sex steroids, which appear to modulate the activities of glutathione-related enzymes. Our experimental observation corroborates previous epidemiological studies showing the greater developmental effects in male than in female humans exposed to MeHg., (Copyright © 2008 Elsevier B.V. All rights reserved.)

The exposure to different kinds of stress impacts on the reactive oxygen species production with potential risk to the integrity of the tissues. Psychological or biological stress is responsible for a significant increase in the oxidative stress markers and also for activation of the antioxidant defense system. In this study, we analyzed the relationships between social stress, humoral immune response and glutathione-related antioxidant defenses. Groups of male Swiss mice were subjected to different lengths of social stress exposure (social confrontation) which varied from 1 up to 13 days. As a biological stressor, 10(9) sheep red blood cells (SRBC)/mL were injected by intraperitoneal route. As controls, animals not subjected to social stress and/or injected with vehicle solution were used. The serum samples and the cerebral cortex were collected at 4 h, 3, 5, 7, 9, 11, and 13 days after the end of social confrontation. The results indicated that the antioxidant enzymes activities were affected by psychological as well as by biological stressor. These alterations were dependent on the timing of stress exposure which resulted in a positive or in a negative correlation between the antibody titres to SRBC and antioxidant enzymes. We also discuss the possible role of SRBC injection in the modulation of the effects of psychosocial stress on antioxidant metabolism.

Brain preconditioning refers to a wide range of treatments that induce a neuronal tolerance state where neuronal tissue become more resistant to a subsequent lethal insult. The mechanisms underlying the preconditioning-induced brain tolerance are not fully understood, but up-regulation of antioxidant enzymes activity has been suggested to play an important role. In order to test this hypothesis, evaluation of glutathione (GSH) scavenger system was carried out in mice showing the neuroprotective effect of NMDA preconditioning against quinolinic acid (QA)-induced seizures. NMDA is known to prevent seizures in 53% of the animals and completely prevent neural damage against QA. Mice were preconditioned by a non-convulsant NMDA dose (75 mg/kg, 10 ml/kg i.p.) 24 h before QA infusion (4 microl, 9.2 mM i.c.v.). GSH content and enzymatic activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) were evaluated in the cerebral cortex and hippocampus 24 h after QA infusion. NMDA preconditioning and QA infusion did not alter GSH content, GR and G6PDH activities, however, an increase in GST activity was observed in the cerebral cortex from mice. Moreover, NMDA pretreatment was able to prevent the QA-induced decrease in hippocampal GPx activity, but it was not effective against the decreased cortical GPx activity. These results indicate that, although NMDA preconditioning and QA toxicity modulate the activity of some GSH related enzymes, GSH metabolism is not directly linked to the neuroprotective effect induced by NMDA preconditioning.

Cipura paludosa (Iridaceae), a native plant widely distributed in the north of Brazil, is used in traditional medicine as an anti-inflammatory and analgesic agent, against tuberculosis and gonorrhoea and for regulation of menstrual flow. However, scientific studies on the pharmacological properties of C. paludosa are scarce. We have examined the potential protective effects of the ethanolic extract of C. paludosa against methyl mercury (MeHg)-induced neurotoxicity in adult mice. MeHg was diluted in drinking water (40 mg/l, freely available) and the ethanolic C. paludosa extract (CE) was diluted in a 150 mM NaCl solution and administered by gavage (10 and 100 mg/kg body weight, respectively, twice a day). Because treatment lasted for 14 days and each animal weighed around 40 g, the total dosage of plant extract given to each mouse was 5.6 and 56 g, respectively. After the treatment period, MeHg exposure induced a significant deficit in the motor coordination, which was evident by a reduction (90%) in the falling latency in the rotarod apparatus. Interestingly, this phenomenon was completely recovered to control levels by CE co-administration, independent of dosages. MeHg exposure inhibited cerebellar glutathione peroxidase (mean percentage inhibition of 42%) - an important enzyme involved in the detoxification of endogenous peroxides - and this effect was prevented by co-administration of CE. Conversely, MeHg exposure increased cerebellar glutathione reductase activity (mean percentage inhibition of 70%), and this phenomenon was not affected by C. paludosa co-administration. Neither MeHg nor CE changed the cerebellar glutathione levels. This study has shown for the first time, the in vivo protective effects of CE against MeHg-induced neurotoxicity. In addition, our findings encourage studies concerning the beneficial effects of C. paludosa on neurological conditions related to excitotoxicity and oxidative stress.