Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O₂^•−) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O₂^•− in mice. However, the pathological contribution of XO-derived O₂^•− production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O₂^•− derived from XOR in Sod1^−/− mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1^−/− mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1^−/− mice. These results showed that XOR-mediated O₂^•− production is relatively uninvolved in the age-related pathologies in Sod1^−/− mice.     

How to Cope with the Challenges of Environmental Stresses in the Era of Global Climate Change: An Update on ROS Stave off in Plants

With the advent of human civilization and anthropogenic activities in the shade of urbanization and global climate change, plants are exposed to a complex set of abiotic stresses. These stresses affect plants’ growth, development, and yield and cause enormous crop losses worldwide. In this alarming scenario of global climate conditions, plants respond to such stresses through a highly balanced and finely tuned interaction between signaling molecules. The abiotic stresses initiate the quick release of reactive oxygen species (ROS) as toxic by-products of altered aerobic metabolism during different stress conditions at the cellular level. ROS includes both free oxygen radicals {superoxide (O₂^•−) and hydroxyl (OH⁻)} as well as non-radicals [hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂)]. ROS can be generated and scavenged in different cell organelles and cytoplasm depending on the type of stimulus. At high concentrations, ROS cause lipid peroxidation, DNA damage, protein oxidation, and necrosis, but at low to moderate concentrations, they play a crucial role as secondary messengers in intracellular signaling cascades. Because of their concentration-dependent dual role, a huge number of molecules tightly control the level of ROS in cells. The plants have evolved antioxidants and scavenging machinery equipped with different enzymes to maintain the equilibrium between the production and detoxification of ROS generated during stress. In this present article, we have focused on current insights on generation and scavenging of ROS during abiotic stresses. Moreover, the article will act as a knowledge base for new and pivotal studies on ROS generation and scavenging.     

Kansuinine A Ameliorates Atherosclerosis and Human Aortic Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Production and Suppressing IKKβ/IκBα/NF-κB Signaling

Reactive oxygen species (ROS)-induced vascular endothelial cell apoptosis is strongly associated with atherosclerosis progression. Herein, we aimed to examine whether Kansuinine A (KA), extracted from Euphorbia kansui L., prevents atherosclerosis development in a mouse model and inhibits cell apoptosis through oxidative stress reduction. Atherosclerosis development was analyzed in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet (HFD) using Oil Red O staining and H&E staining. Human aortic endothelial cells (HAECs) were treated with KA, followed by hydrogen peroxide (H₂O₂), to investigate the KA-mediated inhibition of ROS-induced oxidative stress and cell apoptosis. Oil Red O staining and H&E staining showed that atherosclerotic lesion size was significantly smaller in the aortic arch of ApoE^−/− mice in the HFD+KA group than that in the aortic arch of those in the HFD group. Further, KA (0.1–1.0 μM) blocked the H₂O₂-induced death of HAECs and ROS generation. The H₂O₂-mediated upregulation of phosphorylated IKKβ, phosphorylated IκBα, and phosphorylated NF-κB was suppressed by KA. KA also reduced the Bax/Bcl-2 ratio and cleaved caspase-3 expression, preventing H₂O₂-induced vascular endothelial cell apoptosis. Our results indicate that KA may protect against ROS-induced endothelial cell apoptosis and has considerable clinical potential in the prevention of atherosclerosis and cardiovascular diseases.     

Pathological Relationship between Intracellular Superoxide Metabolism and p53 Signaling in Mice

Intracellular superoxide dismutases (SODs) maintain tissue homeostasis via superoxide metabolism. We previously reported that intracellular reactive oxygen species (ROS), including superoxide accumulation caused by cytoplasmic SOD (SOD1) or mitochondrial SOD (SOD2) insufficiency, induced p53 activation in cells. SOD1 loss also induced several age-related pathological changes associated with increased oxidative molecules in mice. To evaluate the contribution of p53 activation for SOD1 knockout (KO) (Sod1^−/−) mice, we generated SOD1 and p53 KO (double-knockout (DKO)) mice. DKO fibroblasts showed increased cell viability with decreased apoptosis compared with Sod1^−/− fibroblasts. In vivo experiments revealed that p53 insufficiency was not a great contributor to aging-like tissue changes but accelerated tumorigenesis in Sod1^−/− mice. Furthermore, p53 loss failed to improve dilated cardiomyopathy or the survival in heart-specific SOD2 conditional KO mice. These data indicated that p53 regulated ROS-mediated apoptotic cell death and tumorigenesis but not ROS-mediated tissue degeneration in SOD-deficient models.     

Essential Protective Role of Catalytically Active Antibodies (Abzymes) with Redox Antioxidant Functions in Animals and Humans

During the life of aerobic organisms, the oxygen resulting from numerous reactions is converted into reactive oxygen species (ROS). Many ROS are dangerous due to their high reactivity; they are strong oxidants, and react with various cell components, leading to their damage. To protect against ROS overproduction, enzymatic and non-enzymatic systems are evolved in aerobic cells. Several known non-enzymatic antioxidants have a relatively low specific antioxidant activity. Superoxide dismutases, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, and the peroxiredoxin families are the most important enzyme antioxidants. Artificial antibodies catalyzing redox reactions using different approaches have been created. During the past several decades, it has been shown that the blood and various biological fluids of humans and animals contain natural antibodies that catalyze different redox reactions, such as classical enzymes. This review, for the first time, summarizes data on existing non-enzymatic antioxidants, canonical enzymes, and artificial or natural antibodies (abzymes) with redox functions. Comparing abzymes with superoxide dismutase, catalase, peroxide-dependent peroxidase, and H₂O₂-independent oxidoreductase activities with the same activities as classical enzymes was carried out. The features of abzymes with the redox activities are described, including their exceptional diversity in the optimal pH values, dependency and independence on various metal ions, and the reaction rate constants for healthy donors and patients with different autoimmune diseases. The entire body of evidence indicates that abzymes with redox antioxidant activities existing in the blood for a long time compared to enzymes are an essential part of the protection system of humans and animals from oxidative stress.     

Impact of Zinc on Oxidative Signaling Pathways in the Development of Pulmonary Vasoconstriction Induced by Hypobaric Hypoxia

Hypobaric hypoxia is a condition that occurs at high altitudes (>2500 m) where the partial pressure of gases, particularly oxygen (PO₂), decreases. This condition triggers several physiological and molecular responses. One of the principal responses is pulmonary vascular contraction, which seeks to optimize gas exchange under this condition, known as hypoxic pulmonary vasoconstriction (HPV); however, when this physiological response is exacerbated, it contributes to the development of high-altitude pulmonary hypertension (HAPH). Increased levels of zinc (Zn²⁺) and oxidative stress (known as the “ROS hypothesis”) have been demonstrated in the vasoconstriction process. Therefore, the aim of this review is to determine the relationship between molecular pathways associated with altered Zn²⁺ levels and oxidative stress in HPV in hypobaric hypoxic conditions. The results indicate an increased level of Zn²⁺, which is related to increasing mitochondrial ROS (mtROS), alterations in nitric oxide (NO), metallothionein (MT), zinc-regulated, iron-regulated transporter-like protein (ZIP), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced protein kinase C epsilon (PKCε) activation in the development of HPV. In conclusion, there is an association between elevated Zn²⁺ levels and oxidative stress in HPV under different models of hypoxia, which contribute to understanding the molecular mechanism involved in HPV to prevent the development of HAPH.     

Inhibition of NADPH Oxidase by Apocynin Attenuates Progression of Atherosclerosis

Of the multiple sources of reactive oxygen species (ROS) in the blood vessel, NADPH oxidases are the primary source. Whereas several studies have implicated NADPH oxidases in the initiation of atherosclerosis, their roles in disease progression are incompletely understood. Our objective was to determine the potential clinical relevance of inhibiting NADPH oxidase in established atherosclerosis. Using a hypercholesteremic murine model of atherosclerosis (ApoE^−/−/LDLR^−/− (AS) mice on normal chow diet), we first established a time-dependent relationship between superoxide levels and lesion size in AS mice. Next, we identified NADPH oxidase as the primary source of ROS in atherosclerotic lesions. Treatment of aortic segments from AS mice with apocynin, which interferes with NADPH oxidase activation in part by preventing translocation of the subunit p47^phox, significantly reduced superoxide levels. Moreover, addition of apocynin to the drinking water of AS mice produced a decrease in lesion size as compared to untreated AS mice, with the effect most pronounced in the thoracoabdominal aorta but absent from the aortic arch. Granulocyte function in AS+apocynin mice was suppressed, confirming efficacy of apocynin treatment. We conclude that apocynin attenuates the progression of atherosclerosis in hypercholesterolemic mice, potentially by its ability to inhibit generation of superoxide by NADPH oxidase.     

CSH RNA Interference Reduces Global Nutrient Uptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the ³H₂O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.     

Early Inactivation of Membrane Estrogen Receptor Alpha (ERα) Recapitulates the Endothelial Dysfunction of Aged Mouse Resistance Arteries

Flow-mediated dilation (FMD) of resistance arteries is essential for tissue perfusion but it decreases with ageing. As estrogen receptor alpha (Erα encoded by Esr1), and more precisely membrane ERα, plays an important role in FMD in young mice in a ligand-independent fashion, we evaluated its influence on this arteriolar function in ageing. We first confirmed that in young (6-month-old) mice, FMD of mesenteric resistance arteries was reduced in Esr1−/− (lacking ERα) and C451A-ERα (lacking membrane ERα). In old (24-month-old) mice, FMD was reduced in WT mice compared to young mice, whereas it was not further decreased in Esr1−/− and C451A-ERα mice. Markers of oxidative stress were similarly increased in old WT and C451A-ERα mice. Reduction in oxidative stress with superoxide dismutase plus catalase or Mito-tempo, which reduces mitochondrial superoxide restored FMD to a normal control level in young C451A-ERα mice as well as in old WT mice and old C451A-ERα mice. Estradiol-mediated dilation was absent in old WT mice. We conclude that oxidative stress is a key event in the decline of FMD, and that an early defect in membrane ERα recapitulates phenotypically and functionally ageing of these resistance arteries. The loss of this function could take part in vascular ageing.     

Effects of Aging and Hypercholesterolemia on Oxidative Stress and DNA Damage in Bone Marrow Mononuclear Cells in Apolipoprotein E-deficient Mice

Recent evidence from apolipoprotein E-deficient (apoE^−/−) mice shows that aging and atherosclerosis are closely associated with increased oxidative stress and DNA damage in some cells and tissues. However, bone marrow cells, which are physiologically involved in tissue repair have not yet been investigated. In the present study, we evaluated the influence of aging and hypercholesterolemia on oxidative stress, DNA damage and apoptosis in bone marrow cells from young and aged apoE^−/− mice compared with age-matched wild-type C57BL/6 (C57) mice, using the comet assay and flow cytometry. The production of both superoxide and hydrogen peroxide in bone marrow cells was higher in young apoE^−/− mice than in age-matched C57 mice, and reactive oxygen species were increased in aged C57 and apoE^−/− mice. Similar results were observed when we analyzed the DNA damage and apoptosis. Our data showed that both aging and hypercholesterolemia induce the increased production of oxidative stress and consequently DNA damage and apoptosis in bone marrow cells. This study is the first to demonstrate a functionality decrease of the bone marrow, which is a fundamental extra-arterial source of the cells involved in vascular injury repair.     

Pro‐oxidant Properties of AZT and other Thymidine Analogues in Macrophages: Implication of the Azido Moiety in Oxidative Stress

Zidovudine (azidothymidine, AZT) was the first drug approved for human immunodeficiency virus (HIV) treatment. Unfortunately, AZT is known to lead to severe side effects, many of which are generally thought to result from increased reactive oxygen species (ROS) production. In this work, the pro‐oxidative properties of AZT and other thymidine analogues were investigated electrochemically at microelectrodes. Macrophages pre‐incubated with AZT were found to release significant amounts of reactive species, including H₂O₂, ONOO^?, NO^. and NO₂^?. Interestingly, the total amounts of released species were the greatest when cells were incubated with azido‐containing analogues. The pro‐oxidative effect of these compounds decreased significantly when the free azide terminal group was modified by reaction with a triosmium cluster. As expected, thymidine incubation did not lead to any increase in overall ROS levels. This work implicates the azido moiety in AZT‐induced oxidative stress.     

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O₂^•− (and consequently, H₂O₂) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H₂O₂ had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H₂O₂ treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed.     

Oxidative Stress and Chemoradiation-Induced Oral Mucositis: A Scoping Review of In Vitro,In Vivo and Clinical Studies

Chemoradiation-induced mucositis is a debilitating condition of the gastrointestinal tract eventuating from antineoplastic treatment. It is believed to occur primarily due to oxidative stress mechanisms, which generate Reactive Oxygen Species (ROS). The aim of this scoping review was to assess the role of oxidative stress in the development of Oral Mucositis (OM). Studies from the literature, published in MEDLINE and SCOPUS, that evaluated the oxidative stress pathways or antioxidant interventions for OM, were retrieved to elucidate the current understanding of their relationship. Studies failing inclusion criteria were excluded, and those suitable underwent data extraction, using a predefined data extraction table. Eighty-nine articles fulfilled criteria, and these were sub-stratified into models of study (in vitro, in vivo, or clinical) for evaluation. Thirty-five clinical studies evaluated antioxidant interventions on OM’s severity, duration, and pain, amongst other attributes. A number of clinical studies sought to elucidate the protective or therapeutic effects of compounds that had been pre-determined to have antioxidant properties, without directly assessing oxidative stress parameters (these were deemed “indirect evidence”). Forty-seven in vivo studies assessed the capacity of various compounds to prevent OM. Findings were mostly consistent, reporting reduced OM severity associated with a reduction in ROS, malondialdehyde (MDA), myeloperoxidase (MPO), but higher glutathione (GSH) and superoxide dismutase (SOD) activity or expression. Twenty-one in vitro studies assessed potential OM therapeutic interventions. The majority demonstrated successful a reduction in ROS, and in select studies, secondary molecules were assessed to identify the mechanism. In summary, this review highlighted numerous oxidative stress pathways involved in OM pathogenesis, which may inform the development of novel therapeutic targets.     

Oxidative Stress in Ageing and Chronic Degenerative Pathologies: Molecular Mechanisms Involved in Counteracting Oxidative Stress and Chronic Inflammation

Ageing and chronic degenerative pathologies demonstrate the shared characteristics of high bioavailability of reactive oxygen species (ROS) and oxidative stress, chronic/persistent inflammation, glycation, and mitochondrial abnormalities. Excessive ROS production results in nucleic acid and protein destruction, thereby altering the cellular structure and functional outcome. To stabilise increased ROS production and modulate oxidative stress, the human body produces antioxidants, “free radical scavengers”, that inhibit or delay cell damage. Reinforcing the antioxidant defence system and/or counteracting the deleterious repercussions of immoderate reactive oxygen and nitrogen species (RONS) is critical and may curb the progression of ageing and chronic degenerative syndromes. Various therapeutic methods for ROS and oxidative stress reduction have been developed. However, scientific investigations are required to assess their efficacy. In this review, we summarise the interconnected mechanism of oxidative stress and chronic inflammation that contributes to ageing and chronic degenerative pathologies, including neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), cardiovascular diseases CVD, diabetes mellitus (DM), and chronic kidney disease (CKD). We also highlight potential counteractive measures to combat ageing and chronic degenerative diseases.     

Oxidative Stress in the Pathogenesis of Keratoconus and Fuchs Endothelial Corneal Dystrophy

Due to its localization and function, the cornea is regularly exposed to sunlight and atmospheric oxygen, mainly dioxygen, which produce reactive oxygen species (ROS). Therefore, corneal cells are particularly susceptible to oxidative stress. The accumulation of ROS in the cornea may affect signal transduction, proliferation and may also promote cell death. The cornea has several enzymatic and non-enzymatic antioxidants involved in ROS scavenging, but in certain conditions they may not cope with oxidative stress, leading to diseases of the eye. Keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD) are multifactorial diseases of the cornea, in which pathogenesis is not fully understood. However, increased levels of oxidative stress markers detected in these disorders indicate that oxidative stress may play an important role in their development and progression. These markers are: (i) decreased levels of non-enzymatic antioxidants, and (ii) decreased expression of genes encoding antioxidative enzymes, including thioredoxin reductase, peroxiredoxins, superoxide dismutase, glutathione S-transferase, and aldehyde dehydrogenase. Moreover, the FECD endothelium displays higher levels of oxidative DNA damage, especially in mitochondrial DNA (mtDNA), whereas KC cornea shows abnormal levels of some components of oxidative phosphorylation encoded by mtDNA. In this review we present some considerations and results of experiments supporting the thesis on the important role of oxidative stress in KC and FECD pathology.     

Neuroprotective Effects of Erucin against 6-Hydroxydopamine-Induced Oxidative Damage in a Dopaminergic-like Neuroblastoma Cell Line

Oxidative stress (OS) contributes to the cascade leading to the dysfunction or death of dopaminergic neurons during Parkinson’s disease (PD). A strategy to prevent the OS of dopaminergic neurons may be the use of phytochemicals as inducers of endogenous antioxidants and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with isothiocyanate erucin (ER), a compound of cruciferous vegetables, resulted in significant increases of both total glutathione (GSH) levels and total antioxidant capacity at the cytosolic level. The increase of GSH levels was associated with an increase in the resistance of SH-SY5Y cells to neuronal death, in terms of apoptosis, induced by 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with ER was also shown to prevent the redox status impairment, in terms of intracellular ROS and O₂^•− formation, and loss of mitochondrial membrane potential, early events that are initiators of the apoptotic process, induced by 6-OHDA. Last, the antiapoptotic and antioxidant effects of ER were abolished by buthionine sulfoximine, supporting the main role of GSH in the neuroprotective effects recorded by ER. These results suggest that ER may prevent the oxidative damage induced by 6-OHDA.