Expression Of Several Antioxidant Enzymes Research Articles

NRF2 is required for neonatal mouse beta cell growth by maintaining redox balance and promoting mitochondrial biogenesis and function.

All forms of diabetes result from insufficient functional beta cell mass. Due to the relatively limited expression of several antioxidant enzymes, beta cells are highly vulnerable to pathological levels of reactive oxygen species (ROS), which can lead to the reduction of functional beta cell mass. During early postnatal ages, both human and rodent beta cells go through a burst of proliferation that quickly declines with age. The exact mechanisms that account for neonatal beta cell proliferation are understudied but mitochondrial release of moderated ROS levels has been suggested as one of the main drivers. We previously showed that, apart from its conventional role in protecting beta cells from oxidative stress, the nuclear factor erythroid 2-related factor 2 (NRF2) is also essential for beta cell proliferation. We therefore hypothesised that NRF2, which is activated by ROS, plays an essential role in beta cell proliferation at early postnatal ages. Beta cell NRF2 levels and beta cell proliferation were measured in pancreatic sections from non-diabetic human cadaveric donors at different postnatal ages, childhood and adulthood. Pancreatic sections from 1-, 7-, 14- and 28-day-old beta cell-specific Nrf2 (also known as Nfe2l2)-knockout mice (βNrf2KO) or control (Nrf2lox/lox) mice were assessed for beta cell NRF2 levels, beta cell proliferation, beta cell oxidative stress, beta cell death, nuclear beta cell pancreatic duodenal homeobox protein 1 (PDX1) levels and beta cell mass. Seven-day-old βNrf2KO and Nrf2lox/lox mice were injected daily with N-acetylcysteine (NAC) or saline (154 mmol/l NaCl) to explore the potential contribution of oxidative stress to the phenotypes seen in βNrf2KO mice at early postnatal ages. RNA-seq was performed on 7-day-old βNrf2KO and Nrf2lox/lox mice to investigate the mechanisms by which NRF2 stimulates beta cell proliferation at early postnatal ages. Mitochondrial biogenesis and function were determined using dispersed islets from 7-day-old βNrf2KO and Nrf2lox/lox mice by measuring MitoTracker intensity, mtDNA/gDNA ratio and ATP/ADP ratio. To study the effect of neonatal beta cell-specific Nrf2 deletion on glucose homeostasis in adulthood, blood glucose, plasma insulin and insulin secretion were determined and a GTT was performed on 3-month-old βNrf2KO and Nrf2lox/lox mice fed on regular diet (RD) or high-fat diet (HFD). The expression of the master antioxidant regulator NRF2 was increased at early postnatal ages in both human (1 day to 19 months old, 31%) and mouse (7 days old, 57%) beta cells, and gradually declined with age (8% in adult humans, 3.77% in adult mice). A significant correlation (R2=0.568; p=0.001) was found between beta cell proliferation and NRF2 levels in human beta cells. Seven-day-old βNrf2KO mice showed reduced beta cell proliferation (by 65%), beta cell nuclear PDX1 levels (by 23%) and beta cell mass (by 67%), and increased beta cell oxidative stress (threefold) and beta cell death compared with Nrf2lox/lox control mice. NAC injections increased beta cell proliferation in 7-day-old βNrf2KO mice (3.4-fold) compared with saline-injected βNrf2KO mice. Interestingly, RNA-seq of islets isolated from 7-day-old βNrf2KO mice revealed reduced expression of mitochondrial RNA genes and genes involved in the electron transport chain. Islets isolated from 7-day old βNrf2KO mice presented reduced MitoTracker intensity (by 47%), mtDNA/gDNA ratio (by 75%) and ATP/ADP ratio (by 68%) compared with islets from Nrf2lox/lox littermates. Lastly, HFD-fed 3-month-old βNrf2KO male mice displayed a significant reduction in beta cell mass (by 35%), a mild increase in non-fasting blood glucose (1.2-fold), decreased plasma insulin (by 14%), and reduced glucose tolerance (1.3-fold) compared with HFD-fed Nrf2lox/lox mice. Our study highlights NRF2 as an essential transcription factor for maintaining neonatal redox balance, mitochondrial biogenesis and function and beta cell growth, and for preserving functional beta cell mass in adulthood under metabolic stress. Sequencing data are available in the NCBI Gene Expression Omnibus, accession number GSE242718 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242718 ).

Characterization of zearalenone-induced hepatotoxicity and its mechanisms by transcriptomics in zebrafish model

Zearalenone is a mycotoxin produced by several species of Fusarium fungi, which contaminates crop and cereal products worldwide. It is widely distributed and can be transported from agricultural fields to the aquatic environment via soil run-off. Zearalenone exposure can cause serious health problems to humans and animals, including estrogenic, immunotoxic, and xenogenic effects. Though its hepatotoxicity has been reported by few studies, the underlying mechanisms are yet to be investigated. This study aimed to comprehensively evaluate the hepatotoxic effects of zearalenone and its molecular mechanism in the zebrafish model system. First, we found zearalenone exposure can cause liver injury, as evidenced by reduced liver size, decreased liver-specific fluorescence, increased aspartate aminotransferase (AST) activity, delayed yolk sac absorption and lipid accumulation. Then, RNA sequencing (RNA-seq) was performed using dissected zebrafish fry liver, which found genes involved in oxidation and reduction were significantly enriched. Quantitative real-time PCR further confirmed the dysregulated expression of several antioxidant enzymes. Additionally, lipid peroxidation was proved by increased malondialdehyde (MDA) production and gene expression at the mRNA level. In contrast to the previous study, apoptosis was likely decreased in response to zearalenone exposure. Last, glucuronidation and amino acid metabolism were also disrupted by zearalenone. Our results revealed the complex mechanism of zearalenone-induced hepatotoxicity, which is a valuable contribution to a more comprehensive understanding of the toxicity of zearalenone.

Circulating primitive murine erythroblasts undergo complex proteomic and metabolomic changes during terminal maturation

AbstractPrimitive erythropoiesis is a critical component of the fetal cardiovascular network and is essential for the growth and survival of the mammalian embryo. The need to rapidly establish a functional cardiovascular system is met, in part, by the intravascular circulation of primitive erythroid precursors that mature as a single semisynchronous cohort. To better understand the processes that regulate erythroid precursor maturation, we analyzed the proteome, metabolome, and lipidome of primitive erythroblasts isolated from embryonic day (E) 10.5 and E12.5 of mouse gestation, representing their transition from basophilic erythroblast to orthochromatic erythroblast (OrthoE) stages of maturation. Previous transcriptional and biomechanical characterizations of these precursors have highlighted a transition toward the expression of protein elements characteristic of mature red blood cell structure and function. Our analysis confirmed a loss of organelle-specific protein components involved in messenger RNA processing, proteostasis, and metabolism. In parallel, we observed metabolic rewiring toward the pentose phosphate pathway, glycolysis, and the Rapoport-Luebering shunt. Activation of the pentose phosphate pathway in particular may have stemmed from increased expression of hemoglobin chains and band 3, which together control oxygen-dependent metabolic modulation. Increased expression of several antioxidant enzymes also indicated modification to redox homeostasis. In addition, accumulation of oxylipins and cholesteryl esters in primitive OrthoE cells was paralleled by increased transcript levels of the p53-regulated cholesterol transporter (ABCA1) and decreased transcript levels of cholesterol synthetic enzymes. The present study characterizes the extensive metabolic rewiring that occurs in primary embryonic erythroid precursors as they prepare to enucleate and continue circulating without internal organelles.

Lack of Type I Interferon Signaling Ameliorates Respiratory Syncytial Virus-Induced Lung Inflammation and Restores Antioxidant Defenses.

Respiratory syncytial virus (RSV) infection in mouse and human lung is associated with pathogenic inflammation and oxidative injury. RSV impairs antioxidant responses by increasing the degradation of transcription factor NF-E2-related factor 2 (NRF2), which controls the expression of several antioxidant enzymes (AOEs). In addition to its protective effects, type I IFNs have been increasingly recognized as important mediators of host pathogenic responses during acute respiratory viral infections. We used a mouse model of RSV infection to investigate the effect of lack of type I interferon (IFN) receptor on viral-mediated clinical disease, airway inflammation, NRF2 expression, and antioxidant defenses. In the absence of type I IFN signaling, RSV-infected mice showed significantly less body weight loss and airway obstruction, as well as a significant reduction in cytokine and chemokine secretion and airway inflammation. Lack of type I IFN receptor was associated with greatly reduced virus-induced promyelocytic leukemia lung protein expression, which we showed to be necessary for virus-induced NRF2 degradation in a cell model of infection, resulting in restoration of NRF2 levels, AOE expression, and airway antioxidant capacity. Our data support the concept that modulation of type I IFN production and/or signaling could represent an important therapeutic strategy to ameliorate severity of RSV-induced lung disease.

Circulating Primitive Erythroblasts in the Murine Embryo Undergo Complex Proteomic and Metabolomic Changes during Terminal Maturation

Primitive erythropoiesis is a critical component of the fetal cardiovascular network and is essential for growth and survival of the mammalian embryo. The need to rapidly establish a functional cardiovascular system in the post-implantation murine embryo is met, in part, by the intravascular circulation of primitive erythroid precursors that mature as a single semi-synchronous cohort. To better understand the processes that regulate erythroid precursor maturation, we analyzed the proteome, metabolome and lipidome of primitive erythroblasts isolated from embryonic day (E)10.5 and E12.5 of mouse gestation, which represents their transition from basophilic erythroblast (BasoE) to orthochromatic erythroblast (OrthoE) stages of maturation. Previous transcriptional and biomechanical characterization of these precursors has highlighted a transition towards the expression of protein elements characteristic of mature red blood cell structure and function, including initial formation of a functional erythroid-specific cytoskeleton. Our analysis confirmed a loss of organelle-specific protein components - from mRNA maturation to translation, from metabolism to protein degradation - including ribosome, spliceosome, mitochondria and proteasomes. In parallel, we observed a metabolic rewiring towards the pentose phosphate pathway, glycolysis and the Rapoport-Luebering shunt, which is consistent with the progressively decreasing steady state levels of carboxylic acids, acylcarnitines and free fatty acids along with decreased mitochondrial content. In addition, activation of the pentose phosphate pathway in particular may stem from increases in the expression of mature red blood cell components including hemoglobin chains and band 3 that control oxygen-dependent metabolic modulation. Furthermore, this metabolic reprogamming may reflect increased control of oxidative stress in line with increased expression of several antioxidant enzymes, including catalase, peroxiredoxins and glutathione peroxidase 4. On the other hand, accumulation of oxylipins and cholesteryl esters in primitive OrthoE cells was paralleled by increased transcript levels of the p53-regulated cholesterol transporter (ABCA1) and decreased transcript levels of cholesterol synthetic enzymes including mevalonate kinase (MVK) and phosphomevalonate kinase (PVMK), highlighting transcriptional control of metabolic reprogramming during this developmental process. The present study characterizes the extensive metabolic rewiring that occurs in primary embryonic erythroid precursors as they prepare to enucleate and continue circulating without internal organelles. DisclosuresNemkov: Omix Thecnologies: Other: Co-founder. Hansen: Omix Technologies: Other: Co-founder; ScalmiBio: Membership on an entity's Board of Directors or advisory committees. Palis: Rubius Therapeutics: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

An insight into the ameliorative effects of melatonin against chromium induced oxidative stress and DNA damage: a review

Chromium (Cr), a ubiquitous metal, has become a potent pollutant due to global industrialization, leading to pollution of air, water, and food that impacts human health. The most stable forms of Cr are Cr(III) and Cr(VI) (the major product of industrial activities). Cr(III) is a micronutrient essential for maintaining normal blood glucose and lipid profiles in our body but it can also form Cr (III)-DNA adducts. In addition, it directly produces reactive oxygen species (ROS) via Fenton and Haber-Weiss reactions; leading to tissue injuries. Cr (VI) has the capacity to generate Cr(V), Cr (IV), and Cr(III), respectively under suitable conditions. These intermediates also damage to biological macromolecules by interactions with several enzymatic and non-enzymatic antioxidants. For example, Cr(III) can make double DNA strands breaking to inhibit DNA replication, induce DNA oxidation, and DNA adducts formation. All of these lead to the development of malignancy. Melatonin, a potent radical scavenger as well as a metal chelator, effectively chelates Cr(VI) and prevents DNA oxidative damage. Melatonin can upregulate the gene expression of several antioxidant enzymes, and thereby, maintains cellular integrity from the oxidative stress. Thus, melatonin can be a prime molecule to protect against Cr(VI) induced cytotoxicity and genotoxicity. This review aims to highlight the potential benefits of melatonin on Cr(VI) induced oxidative stress and DNA damage.

Contribution of the Nrf2 Pathway on Oxidative Damage and Mitochondrial Failure in Parkinson and Alzheimer's Disease.

The increase in human life expectancy has become a challenge to reduce the deleterious consequences of aging. Nowadays, an increasing number of the population suffer from age-associated neurodegenerative diseases including Parkinson’s disease (PD) and Alzheimer’s disease (AD). These disorders present different signs of neurodegeneration such as mitochondrial dysfunction, inflammation, and oxidative stress. Accumulative evidence suggests that the transcriptional factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) plays a vital defensive role orchestrating the antioxidant response in the brain. Nrf2 activation promotes the expression of several antioxidant enzymes that exert cytoprotective effects against oxidative damage and mitochondrial impairment. In this context, several studies have proposed a role of Nrf2 in the pathogenesis of PD and AD. Thus, we consider it important to summarize the ongoing literature related to the effects of the Nrf2 pathway in the context of these diseases. Therefore, in this review, we discuss the mechanisms involved in Nrf2 activity and its connection with mitochondria, energy supply, and antioxidant response in the brain. Furthermore, we will lead our discussion to identify the participation of the Nrf2 pathway in mitochondrial impairment and neurodegeneration present in PD and AD. Finally, we will discuss the therapeutic effects that the Nrf2 pathway activation could have on the cognitive impairment, neurodegeneration, and mitochondrial failure present in PD and AD.

Pterostilbene Protects Against Lipopolysaccharide/D-Galactosamine-Induced Acute Liver Failure by Upregulating the Nrf2 Pathway and Inhibiting NF-κB, MAPK, and NLRP3 Inflammasome Activation.

The purpose of this study was to evaluate the protective effect of pterostilbene (Psb) against lipopolysaccharide and D-galactosamine (L/D)-induced acute liver failure (ALF) in mice and its potential mechanisms. Histology of liver was detected by H&E staining. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in serum and malondialdehyde (MDA), myeloperoxidase (MPO), glutathione (GSH), and superoxide dismutase (SOD) contents in liver were examined using detection kits. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) secretion were detected by ELISA. Meanwhile, MAPK, NF-κB, NLRP3 inflammasome, and Nrf2 were assessed by western blotting. Our findings showed that pretreatment with Psb protected against L/D-induced ALF by lowering the lethality, improving liver histology, reducing ALT, AST, IL-6, IL-1β, TNF-α, MDA, and MPO levels, and boosting liver GSH content and SOD activity. Moreover, Psb pretreatment effectively suppressed inflammation by decreasing NLRP3 inflammasome, MAPK, and NF-κB pathway activations. Moreover, Psb pretreatment efficiently enhanced the expression of several antioxidant enzymes, mainly depending on Nrf2 activation. This was the first study to demonstrate that Psb protects against L/D-induced ALF by inactivating MAPK, NF-κb, and NLRP3 inflammasome and upregulating the Nrf2 signaling pathway, indicating a potential therapeutic application for ALF treatment.

Renal Iron Accumulation and Oxidative Injury With Aging: Effects of Treatment With an Iron Chelator.

Dysregulation of iron metabolism in the kidney may contribute to age-related increases in renal oxidative stress and dysfunction. This study assessed the effects of short-term iron chelation on markers of iron status, oxidative stress, inflammation, and autophagy in the kidneys of old rats. Old Fischer 344 rats (24 months) were treated with deferoxamine (DFO; 200 mg/kg, twice daily for 4.5 days); saline-treated young (6 months) and old rats served as controls. Renal nonheme iron was significantly higher in the old rats, with iron localized in the renal cortex. Ferritin levels were elevated in the kidneys of old rats, while expression of several antioxidant enzymes and mitochondrial proteins were reduced and protein carbonyls increased compared to young rats. DFO treatment significantly reduced ferritin levels, and increased transferrin receptor-1 protein, but did not affect nonheme iron content or protein carbonyls, nor did it reverse age-related changes in antioxidant enzymes and mitochondrial proteins. Although short-term DFO treatment did not mitigate the age-related increase in iron content and oxidative damage, this work demonstrates that old rats respond appropriately to DFO, suggesting that optimization of iron chelation regimens could be useful in improving renal homeostasis with aging.

Transcriptome analysis of genes and metabolic pathways associated with nicotine degradation in Aspergillus oryzae 112822

BackgroundNicotine-degrading microorganisms (NDMs) have recently received much attention since they can consume nicotine as carbon and nitrogen source for growth. In our previous work, we isolated an efficient nicotine-degrading fungus Aspergillus oryzae 112822 and first proposed a novel demethylation pathway of nicotine degradation in fungi. However, the underlying mechanisms of the demethylation pathway remain unresolved. In the present study, we performed a comparative transcriptome analysis to elucidate the molecular mechanisms of nicotine tolerance and degradation in A. oryzae 112822.ResultsWe acquired a global view of the transcriptional regulation of A. oryzae 112822 exposed to nicotine and identified 4381 differentially expressed genes (DEGs) by nicotine treatment. Candidate genes encoding cytochrome P450 monooxygenases (CYPs), FAD-containing amine oxidase, molybdenum cofactor (Moco)-containing hydroxylase, and NADH-dependent and FAD-containing hydroxylase were proposed to participate in the demethylation pathway of nicotine degradation. Analysis of these data also revealed that increased energy was invested to drive nicotine detoxification. Nicotine treatment led to overproduction of reactive oxygen species (ROS), which formed intracellular oxidative stress that could induce the expression of several antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx). Thioredoxin system was induced to restore the intracellular redox homeostasis. Several glutathione S-transferases (GSTs) were induced, most likely to participate in phase II detoxification of nicotine by catalyzing the conjugation of glutathione (GSH) to active metabolites. The toxin efflux pumps, such as the ATP-Binding Cassette (ABC) transporters and the major facilitator superfamily (MFS) transporters, were overexpressed to overcome the intracellular toxin accumulation. By contrast, the metabolic pathways related to cellular growth and reproduction, such as ribosome biogenesis and DNA replication, were inhibited by nicotine treatment.ConclusionThese results revealed that complex regulation networks, involving detoxification, transport, and oxidative stress response accompanied by increased energy investment, were developed for nicotine tolerance and degradation in A. oryzae 112822. This work provided the first insight into the metabolic regulation of nicotine degradation and laid the foundation for further revealing the molecular mechanisms of the nicotine demethylation pathway in filamentous fungi.

Corilagin alleviates acetaminophen-induced hepatotoxicity via enhancing the AMPK/GSK3\u03b2-Nrf2 signaling pathway

BackgroundAcetaminophen (APAP) overdose-induced acute liver failure (ALF) is mainly resulted from uncontrolled oxidative stress. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a key antioxidant transcription factor, is essential for alleviating APAP-induced hepatotoxicity. Corilagin (Cori) is a natural polyphenol compound that possesses effective antioxidant activity; however, the protective effect of Cori on APAP-induced hepatotoxicity is still unknown. The current study aimed to explore whether Cori could mitigate hepatotoxicity caused by APAP and the underlying molecular mechanisms of action.MethodsCell counting kit-8 (CCK-8) assays, Western blotting analysis, dual-luciferase reporter assays, a mouse model, CRISPR/Cas9 knockout technology, and hematoxylin-eosin (H & E) staining were employed to explore the mechanisms by which Cori exerts a protective effect on hepatotoxicity in HepG2 cells and in a mouse model.ResultsOur findings suggested that Cori efficiently decreased APAP-triggered the generation of reactive oxygen species (ROS) and cell death in HepG2 cells. Additionally, Cori significantly induced the expression of several antioxidant enzymes, and this induced expression was closely linked to the upregulation of Nrf2, inhibition of Keap1 protein expression, and promotion of antioxidant response element (ARE) activity in HepG2 cells. Moreover, Cori clearly induced the phosphorylation of AMP-activated protein kinase (AMPK), glycogen synthase kinase-3β (GSK3β), liver kinase B1 (LKB1) and acetyl-CoA carboxylase (ACC). Furthermore, Cori-mediated GSK3β inactivation, Nrf2 upregulation and cytoprotection were abolished by an AMPK inhibitor (Compound C) in HepG2 cells. Lastly, we found that Cori inhibited APAP-induced hepatotoxicity and mediated the expression of many antioxidant enzymes; these results were reversed in Nrf2 −/− HepG2 cells. In vivo, Cori significantly protected against APAP-induced ALF by reducing mortality and alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, attenuating histopathological liver changes, inhibiting myeloperoxidase (MPO) and malondialdehyde (MDA) levels, and increasing the superoxide dismutase (SOD) content and GSH-to-GSSG ratio as well as suppressing c-jun N-terminal kinase (JNK) phosphorylation. However, Cori-induced reductions in mortality, AST and ALT levels, and histopathological liver changes induced by APAP were clearly abrogated in Nrf2-deficienct mice.ConclusionsThese findings principally indicated that Cori effectively protects against APAP-induced ALF via the upregulation of the AMPK/GSK3β-Nrf2 signaling pathway.

Calcitriol inhibits ROS-NLRP3-IL-1β signaling axis via activation of Nrf2-antioxidant signaling in hyperosmotic stress stimulated human corneal epithelial cells

PurposeThe activation of ROS-NLRP3-IL-1β signaling axis induced by hyperosmotic stress (HS) has been recognized as a key priming stage of epithelial inflammation in dry eye pathogenesis. The current study aims to investigate whether calcitriol, the active metabolite of vitamin D3, could protect cells against HS-induced inflammation through modulating this critical step. MethodsHuman corneal epithelial cells (iHCECs) were cultured in hyperosmotic medium (450 mOsM) with various concentrations of calcitriol. Small interfering RNA (siRNA) was used to knock down the expression of vitamin D receptor (VDR) in iHCECs. NLRP3 activation and IL-1β generation were detected by RT-qPCR or ELISA, respectively. Oxidative stress markers including ROS and 8-OHdG were examined by fluorometric analysis. The nuclear translocation of NRF2 was assessed by western blotting. ResultsCalcitriol could protect cells against HS-induced injury through inhibiting ROS-NLRP3-IL-1β signaling axis. Calcitriol remarkably suppressed the expression of NLRP3 inflammasome related genes and the production of IL-1β in cells that were exposed to HS. It could also significantly attenuate HS-induced oxidative stress, shown as the reduced intracellular ROS generation and 8-OHdG staining cells after calcitriol treatment. Calcitriol induced the translocation of NRF2 to the nucleus, and thereby triggered the expression of several antioxidant enzymes. ConclusionThe current study indicated that calcitriol could inhibit the priming stage of HS-induced cellular inflammation, highlighting its potential capacity to prevent and mitigate dry eye related corneal inflammation at an earlier stage.

Dopaminergic dysregulation and impaired associative learning behavior in zebrafish during chronic dietary exposure to selenium

A growing body of evidence indicates that exposure to selenium (Se) can cause neurotoxicity, and this can occur because of its interference with several neurotransmitter systems in humans and animals. Dopamine is a critical modulator of a variety of brain functions and a prime target for environmental neurotoxicants. However, effects of environmentally relevant concentrations of Se on dopaminergic system and its neurobehavioral effects are still largely unknown. For this purpose, we exposed zebrafish, a model organism, to different concentrations of dietary l-selenomethionine (control, 3.5, 11.1, 27.4, and 63.4 μg Se/g dry weight) for a period of 60 days. Cognitive performance of fish was evaluated using a plus maze associative learning paradigm. Oxidative stress, as the main driver of Se neurotoxicity, was assessed by measuring the ratio of reduced to oxidized glutathione (GSH:GSSG), lipid peroxidation (LPO) levels, and mRNA expression of several antioxidant enzymes in the zebrafish brain. Dopamine levels in the brain and the expression of genes involved in dopamine synthesis, storage, reuptake, metabolism, and receptor activation were examined. Moreover, transcription of several synaptic plasticity-related immediate-early and late response genes was determined. Overall, fish fed with the two highest concentrations of dietary Se displayed impaired associative learning. Se exposure also induced oxidative stress in the zebrafish brain, as indicated by a reduction in GSH:GSSG ratio, increased LPO levels, and up-regulation of antioxidant genes in fish treated with the two highest concentrations of Se. An increase in brain dopamine levels associated with altered expression of dopaminergic cell markers was evident in different treatment groups. Moreover, Se exposure led to the down-regulation of immediate-early and late response genes in fish that exhibiting learning impairment. Taken together, the results of this study imply that the induction of oxidative stress and dysregulation of dopaminergic neurotransmission may underlie Se-induced impairment of associative learning in zebrafish.

Effects of low-dose rate γ-irradiation combined with simulated microgravity on markers of oxidative stress, DNA methylation potential, and remodeling in the mouse heart.

PurposeSpace travel is associated with an exposure to low-dose rate ionizing radiation and the microgravity environment, both of which may lead to impairments in cardiac function. We used a mouse model to determine short- and long-term cardiac effects to simulated microgravity (hindlimb unloading; HU), continuous low-dose rate γ-irradiation, or a combination of HU and low-dose rate γ-irradiation.MethodsCardiac tissue was obtained from female, C57BL/6J mice 7 days, 1 month, 4 months, and 9 months following the completion of a 21 day exposure to HU or a 21 day exposure to low-dose rate γ-irradiation (average dose rate of 0.01 cGy/h to a total of 0.04 Gy), or a 21 day simultaneous exposure to HU and low-dose rate γ-irradiation. Immunoblot analysis, rt-PCR, high-performance liquid chromatography, and histology were used to assess inflammatory cell infiltration, cardiac remodeling, oxidative stress, and the methylation potential of cardiac tissue in 3 to 6 animals per group.ResultsThe combination of HU and γ-irradiation demonstrated the strongest increase in reduced to oxidized glutathione ratios 7 days and 1 month after treatment, but a difference was no longer apparent after 9 months. On the other hand, no significant changes in 4-hydroxynonenal adducts was seen in any of the groups, at the measured endpoints. While manganese superoxide dismutase protein levels decreased 9 months after low-dose γ-radiation, no changes were observed in expression of catalase or Nrf2, a transcription factor that determines the expression of several antioxidant enzymes, at the measured endpoints. Inflammatory marker, CD-2 protein content was significantly decreased in all groups 4 months after treatment. No significant differences were observed in α-smooth muscle cell actin protein content, collagen type III protein content or % total collagen.ConclusionsThis study has provided the first and relatively broad analysis of small molecule and protein markers of oxidative stress, T-lymphocyte infiltration, and cardiac remodeling in response to HU with simultaneous exposure to low-dose rate γ-radiation. Results from the late observation time points suggest that the hearts had mostly recovered from these two experimental conditions. However, further research is needed with larger numbers of animals for a more robust statistical power to fully characterize the early and late effects of simulated microgravity combined with exposure to low-dose rate ionizing radiation on the heart.

Humanin Protects RPE Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Upregulation of Mitochondrial Glutathione.

Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1–20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.

Toxic effect of palladium on embryonic development of zebrafish

Since palladium (Pd) is now increasingly used in modern industry, it progressively accumulates in the environment, especially in aquatic ecosystem. The potential toxicity of Pd has therefore caused extensive concern worldwidely. In the present study, we investigated the toxic effect of Pd on zebrafish development. Acute Pd exposure significantly decreased both the survival rate (LC50: 292.6μg/L, viz. 2.75μM) and hatching rate (IC50: 181.5μg/L, viz. 1.71μM) of zebrafish during embryonic development. The most common developmental defect observed in Pd treated embryos is pericardiac edema, which occurs in a dose-dependent manner. Whole mount immunostaining and histological studies revealed that Pd exposure would produce the elongated, string-like heart. The heartbeat rate of zebrafish embryos was also decreased after Pd exposure. Consistently, mRNA expression levels of several cardiac-related genes were affected by Pd, suggesting a potential molecular mechanism of Pd-induced cardiac malformation of zebrafish embryo. Moreover, similar to other metals, Pd exposure resulted in the elevated expression of general metal-inducible genes. It was also found that the expression of several antioxidant enzymes was significantly down-regulated in the presence of Pd. Taken together, our study investigated the effects of Pd on zebrafish embryonic development and its potential molecular mechanisms, paving the way for the full understanding of Pd toxicity.

Natural daucane esters induces apoptosis in leukaemic cells through ROS production

Continuing our research on antiproliferative agents from plants, we extended our interest on further compounds isolated from Ferula communis and Ferulago campestris. One new daucane (DE-20) and one new phenol derivative (PH-3) were isolated and characterized in addition to six daucane, three coumarins and four simple phenolics. The cytotoxic activity was evaluated against a panel of six human tumor cell lines. The derivative DE-17 that resulted moderately active on all the studied cell lines was studied to evaluate its possible mechanism of action. DE-17 was able to induce apoptosis in a time and concentration-dependent manner in SEM and Jurkat cell lines. We observed that DE-17 just after 1h of treatment increased the reactive oxygen species (ROS) production and that the co-incubation of DE-17 with ROS scavengers significantly increased cell viability suggesting that ROS-mediated downstream signaling is essential for the antiproliferative effects of DE-17. At later times of incubation DE-17 induced mitochondrial depolarization, as well as caspase-3 and -9 activation suggesting that apoptosis follow the mitochondrial pathway. Concomitantly to ROS induction, a remarkable decrease of mRNA expression of several antioxidant enzymes and intracellular GSH content was detected in treated cells compared to controls further indicative of oxidative stress. Taken together our results showed for the first time that daucane esters induces apoptotic cell death through a ROS-mediated mechanism in human leukemia cells.

Targeting catalase but not peroxiredoxins enhances arsenic trioxide-induced apoptosis in K562 cells.

Despite considerable efficacy of arsenic trioxide (As2O3) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As2O3 treatment. However, the underlying mechanism is not well understood. Here we show that relative As2O3-resistant K562 cells have significantly lower ROS levels than As2O3-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As2O3. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As2O3. On the contrary, knockdown of catalase markedly enhanced As2O3-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As2O3 in CML treatment.

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures:No relevant conflicts of interest to declare.

The accumulation of endogenous proline induces changes in gene expression of several antioxidant enzymes in leaves of transgenic Swingle citrumelo

Plant exposure to abiotic stresses leads to an accumulation of reactive oxygen species with the concomitant increase in antioxidant defense mechanisms. Previous studies showed that exogenous application of proline mitigate the deleterious effects caused by oxidative stress due to its ability to increase the activity of antioxidant enzymes. However, there are no reports of the effects of high endogenous accumulation of proline in the transcriptional pattern of antioxidant enzymes genes under normal conditions of water supply or in response to water deficit. Here, we show that isoforms of four antioxidant enzymes genes (Ascorbate peroxidase-APX, Catalase-CAT, Superoxide dismutase-SOD and Glutathione reductase-GR) were differentially regulated in leaves of Swingle citrumelo transgenic plants with high endogenous proline accumulation submitted to water deficits and also under normal water supply condition. Proline per se caused a two-fold change in the transcription activity of APX1, APXcl, CAT2 and Cu/ZnSOD2, while during water deficit proline influenced mRNAs levels in APXs and Cu/ZnSODs isoforms, MnSODmit and GRcl. This study adds new information on the role of proline during drought conditions and, more important, without the potential confounding effects imposed by water deficiency. We showed that, in addition to its known effects on diverse plant physiological and biochemical processes, high endogenous proline can also acts as a regulatory/signalling molecule capable of altering the transcript levels of stress-related genes.