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Journal of Biomolecular Screening Apr 2013Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium (DHE) may be...
Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium (DHE) may be unspecific because of overlapping fluorescence of the superoxide-specific product, 2-OH-ethidium (2OH-E), and the unspecific oxidation product, ethidium. Here, we show a new optimized fluorescence spectroscopy protocol that allows rapid and specific detection of superoxide in cell-free systems and intact cells using DHE. We defined new optimized fluorescent settings to measure the superoxide-specific product and minimize the interference of unspecific DHE oxidation products. Using this protocol, we studied real-time superoxide production by xanthine oxidase- and menadione-treated cultured cells. Specificity of the plate reader-based superoxide measurements was confirmed by the inhibition of fluorescence with superoxide dismutase and high-performance liquid chromatography (HPLC) analysis. We show that limitations of the HPLC-based analysis can be overcome by the optimized fluorescence spectroscopy.
Topics: Cells, Cultured; Endothelial Cells; Ethidium; Humans; Spectrometry, Fluorescence; Superoxides; Xanthine Oxidase
PubMed: 23190737
DOI: 10.1177/1087057112468765 -
International Journal of Molecular... May 2023Our study proposes a pharmacological strategy to target cancerous mitochondria via redox-cycling "mitocans" such as quinone/ascorbate (Q/A) redox-pairs, which makes...
Our study proposes a pharmacological strategy to target cancerous mitochondria via redox-cycling "mitocans" such as quinone/ascorbate (Q/A) redox-pairs, which makes cancer cells fragile and sensitive without adverse effects on normal cells and tissues. Eleven Q/A redox-pairs were tested on cultured cells and cancer-bearing mice. The following parameters were analyzed: cell proliferation/viability, mitochondrial superoxide, steady-state ATP, tissue redox-state, tumor-associated NADH oxidase (tNOX) expression, tumor growth, and survival. Q/A redox-pairs containing unprenylated quinones exhibited strong dose-dependent antiproliferative and cytotoxic effects on cancer cells, accompanied by overproduction of mitochondrial superoxide and accelerated ATP depletion. In normal cells, the same redox-pairs did not significantly affect the viability and energy homeostasis, but induced mild mitochondrial oxidative stress, which is well tolerated. Benzoquinone/ascorbate redox-pairs were more effective than naphthoquinone/ascorbate, with coenzyme Q0/ascorbate exhibiting the most pronounced anticancer effects in vitro and in vivo. Targeted anticancer effects of Q/A redox-pairs and their tolerance to normal cells and tissues are attributed to: (i) downregulation of quinone prenylation in cancer, leading to increased mitochondrial production of semiquinone and, consequently, superoxide; (ii) specific and accelerated redox-cycling of unprenylated quinones and ascorbate mainly in the impaired cancerous mitochondria due to their redox imbalance; and (iii) downregulation of tNOX.
Topics: Mice; Animals; Superoxides; Oxidation-Reduction; Ascorbic Acid; Quinones; Neoplasms; Adenosine Triphosphate
PubMed: 37176145
DOI: 10.3390/ijms24098435 -
Biogerontology Oct 2023L-lactate is a catabolite from the anaerobic metabolism of glucose, which plays a paramount role as a signaling molecule in various steps of the cell survival. Its... (Review)
Review
L-lactate is a catabolite from the anaerobic metabolism of glucose, which plays a paramount role as a signaling molecule in various steps of the cell survival. Its activity, as a master tuner of many mechanisms underlying the aging process, for example in the skin, is still presumptive, however its crucial position in the complex cross-talk between mitochondria and the process of cell survival, should suggest that L-lactate may be not a simple waste product but a fine regulator of the aging/survival machinery, probably via mito-hormesis. Actually, emerging evidence is highlighting that ROS are crucial in the signaling of skin health, including mechanisms underlying wound repair, renewal and aging. The ROS, including superoxide anion, hydrogen peroxide, and nitric oxide, play both beneficial and detrimental roles depending upon their levels and cellular microenvironment. Physiological ROS levels are essential for cutaneous health and the wound repair process. Aberrant redox signaling activity drives chronic skin disease in elderly. On the contrary, impaired redox modulation, due to enhanced ROS generation and/or reduced levels of antioxidant defense, suppresses wound healing via promoting lymphatic/vascular endothelial cell apoptosis and death. This review tries to elucidate this issue.
Topics: Humans; Aged; Reactive Oxygen Species; Skin Aging; Superoxides; Oxidation-Reduction; Lactates
PubMed: 36708434
DOI: 10.1007/s10522-023-10018-1 -
Journal of Orthopaedic Research :... Jan 2020Rotator cuff degeneration is one of the factors contributing to rotator cuff tears. Oxidative stress is involved in tendon degeneration, and superoxide-induced oxidative...
Rotator cuff degeneration is one of the factors contributing to rotator cuff tears. Oxidative stress is involved in tendon degeneration, and superoxide-induced oxidative stress plays a pathological role in regulating the balance between oxidation and reduction. The role of oxidative stress in rotator cuff tears, however, is unclear. This study, therefore, aimed to investigate the contribution of superoxide-induced oxidative stress to rotator cuff tears. Seventy patients were recruited and divided into two groups: patients with (Ruptured group) and those without (Unruptured group) a rotator cuff tear. Specimens from both groups were collected during surgery. Degeneration morphology was classified according to the degeneration score. Superoxide-induced oxidative stress was assessed according to dihydroethidium (DHE) relative fluorescence intensity, capacity for antioxidation according to superoxide dismutase (SOD) activity, and the balance between oxidation and reduction based on the redox ratio. Data were compared between groups. Correlations between the degeneration score and the oxidative stress factors were calculated. Degeneration score and DHE relative fluorescence intensity were significantly higher in the Ruptured than the Unruptured group. The SOD activity was not significantly different between groups. Degeneration score was positively correlated with both DHE relative fluorescence intensity and SOD activity. Thus, superoxide-induced oxidative stress and tendon degeneration were greater in rotator cuff tear tissues than in those with no tear, suggesting that oxidative imbalance may be involved in degenerative rotator cuff tears. Clinical Relevance: Understanding the mechanisms of superoxide-induced oxidative stress may lead to targeted tissue therapy for degenerative rotator cuff tears. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:212-218, 2020.
Topics: Adult; Aged; Aged, 80 and over; Ethidium; Female; Fluorescence; Humans; Male; Middle Aged; Oxidative Stress; Retrospective Studies; Rotator Cuff Injuries; Superoxide Dismutase; Superoxides
PubMed: 31520427
DOI: 10.1002/jor.24472 -
Inorganic Chemistry May 2021Superoxide (O) is a toxic radical, generated via the adventitious reduction of dioxygen (O), which has been implicated in a number of human disease states. Nonheme iron...
Superoxide (O) is a toxic radical, generated via the adventitious reduction of dioxygen (O), which has been implicated in a number of human disease states. Nonheme iron enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD), detoxify O via reduction to afford HO and disproportionation to afford O and HO, respectively. The former contains a thiolate in the coordination sphere, which has been proposed to prevent O oxidation to O. The work described herein shows that, in contrast to this, oxidized thiolate-ligated [Fe(SN(tren)(THF)] () is capable of oxidizing O to O. Coordinating anions, Cl and OAc, are shown to inhibit dioxygen evolution, implicating an inner-sphere mechanism. Previously we showed that the reduced thiolate-ligated [Fe(SN(tren))] () is capable of reducing O via a proton-dependent inner-sphere mechanism involving a transient Fe(III)-OOH intermediate. A transient ferric-superoxo intermediate, [Fe(SN(tren))(O)] (), is detected by electronic absorption spectroscopy at -130 °C in the reaction between and KO and shown to evolve O upon slight warming to -115 °C. The DFT calculated O-O (1.306 Å) and Fe-O (1.943 Å) bond lengths of are typical of ferric-superoxo complexes, and the time-dependent DFT calculated electronic absorption spectrum of reproduces the experimental spectrum. The electronic structure of is shown to consist of two antiferromagnetically coupled ( = -180 cm) unpaired electrons, one in a superoxo π*(O-O) orbital and the other in an antibonding π*(Fe(d)-S(p)) orbital.
Topics: Anions; Ferric Compounds; Hydrogen-Ion Concentration; Molecular Conformation; Oxidation-Reduction; Oxygen; Sulfhydryl Compounds; Superoxides
PubMed: 33900756
DOI: 10.1021/acs.inorgchem.1c00336 -
Methods (San Diego, Calif.) Oct 2016Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and...
Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP targeted to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions.
Topics: Animals; Bacterial Proteins; Biological Transport; Biosensing Techniques; Humans; Luminescent Proteins; Microscopy, Confocal; Mitochondria; Reactive Oxygen Species; Signal Transduction; Superoxides
PubMed: 27288722
DOI: 10.1016/j.ymeth.2016.06.004 -
Redox Biology May 2018Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS... (Review)
Review
Teaching the basics of reactive oxygen species and their relevance to cancer biology: Mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies.
Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS generation, the mitochondria and the NADPH oxidase family of enzymes are possibly the most prevalent intracellular sources. In this article, we discuss the methodologies to detect mitochondria-derived superoxide and hydrogen peroxide using conventional probes as well as newly developed assays and probes, and the necessity of characterizing the diagnostic marker products with HPLC and LC-MS in order to rigorously identify the oxidizing species. The redox signaling roles of mitochondrial ROS, mitochondrial thiol peroxidases, and transcription factors in response to mitochondria-targeted drugs are highlighted. ROS generation and ROS detoxification in drug-resistant cancer cells and the relationship to metabolic reprogramming are discussed. Understanding the subtle role of ROS in redox signaling and in tumor proliferation, progression, and metastasis as well as the molecular and cellular mechanisms (e.g., autophagy) could help in the development of combination therapies. The paradoxical aspects of antioxidants in cancer treatment are highlighted in relation to the ROS mechanisms in normal and cancer cells. Finally, the potential uses of newly synthesized exomarker probes for in vivo superoxide and hydrogen peroxide detection and the low-temperature electron paramagnetic resonance technique for monitoring oxidant production in tumor tissues are discussed.
Topics: Humans; Hydrogen Peroxide; Mitochondria; Molecular Targeted Therapy; NADPH Oxidases; Neoplasms; Oxidation-Reduction; Reactive Oxygen Species; Superoxides
PubMed: 29306792
DOI: 10.1016/j.redox.2017.12.012 -
The Journal of Biological Chemistry May 1982Catalase was inhibited by a flux of O2- generated in situ by the aerobic xanthine oxidase reaction. Two distinct types of inhibition could be distinguished. One of these...
Catalase was inhibited by a flux of O2- generated in situ by the aerobic xanthine oxidase reaction. Two distinct types of inhibition could be distinguished. One of these was rapidly established and could be as rapidly reversed by the addition of superoxide dismutase. The second developed slowly and was reversed by ethanol, but not by superoxide dismutase. The rapid inhibition was probably due to conversion of catalase to the ferrooxy state (compound III), while the slow inhibition was due to conversion to the ferryl state (compound II). Since neither compound III nor compound II occurs in the catalatic reaction pathway, they are inactive. This inhibition of catalase by O2- provides the basis for a synergism between superoxide dismutase and catalase. Such synergisms have been observed in vitro and may be significant in vivo.
Topics: Acetaldehyde; Aerobiosis; Binding, Competitive; Catalase; Escherichia coli; Ethanol; Hydrogen-Ion Concentration; Oxygen; Superoxides; Xanthine Oxidase
PubMed: 6279612
DOI: No ID Found -
Plant Signaling & Behavior Feb 2021Cutting is a frequently used model to study the process of adventitious root formation, and excision of cuttings leads to rapid wound response signaling. We recently...
Cutting is a frequently used model to study the process of adventitious root formation, and excision of cuttings leads to rapid wound response signaling. We recently showed that as a wound signal, reactive oxygen species (ROS, mainly hydrogen peroxide) participate in adventitious root induction of hypocotyl cuttings through regulation of auxin biosynthesis and transport. Here, superoxide anion (O), an early type of ROS, exhibited rapid burst at the cutting site immediately in response to wounding in Arabidopsis hypocotyl cuttings. Diphenylene iodonium chloride (DPI, inhibitor of NADPH oxidase) overwhelmingly suppressed O propagation through the hypocotyl. Compared to wild type, O burst only occur in cut base, and upward transduction were inhibited completely in NADPH oxidase mutant . These results indicate O generation and propagation in response to wound and via NADPH oxidase in adventitious root induction of hypocotyl cuttings.
Topics: Arabidopsis; Onium Compounds; Plant Roots; Reactive Oxygen Species; Superoxides
PubMed: 33210579
DOI: 10.1080/15592324.2020.1848086 -
The Biochemical Journal Nov 2018l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation...
l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (HO), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, O). Products were monitored electrophoretically. quenched HO far more effectively than superoxide, but the main products in both cases were 4--oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. HO, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical OH yield; it yielded oxalate, threonate, and OxT. O had no effect on DHA. was oxidatively decarboxylated by HO, Fenton mixture, and O, forming a newly characterised product, 2-oxo-l--pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged HO treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by HO, Fenton mixture, or O also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by HO and superoxide; DKG more readily by O The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting .
Topics: 2,3-Diketogulonic Acid; Ascorbic Acid; Dehydroascorbic Acid; Hydrogen Peroxide; Iron; Models, Chemical; Molecular Structure; Oxidants; Oxidation-Reduction; Reactive Oxygen Species; Superoxides
PubMed: 30348642
DOI: 10.1042/BCJ20180688