Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt₂)₂ was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728 nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5 min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.     

Novel Iron Complexes Behave Like Superoxide Dismutase In Vivo

Novel iron and copper complexes having tris[N-(5-methyl-2-pyridylmethyl)-2-aminoethyl]amine (5MeT-PAA), tris[N-(3-methyl-2-pyridylmethyl)-2-aminoethyl]amine(3MeTPAA),rris[N-(5-methoxycarbonyl-2-pyridylmethyl)-2-aminoethyl]amine (TNAA), tris[(2-thienylmethyI)-2-aminoethyl]amine (TTAA), tris[(2-furylniethyl)-2-aminoethyl]amine (TFAA) or tris[(2-imidazolyl)-2-aminoethyl]amine (TIAA) as ligand. were synthesized to examine the superoxide dismutase (SOD) activity. The concentrations of Fe-3MeTPAA and Fe-TIAA equivalent to 1 unit of SOD (IC₅₀) were 0.5 μM and I.O μM. respectively. Fe-3MeTPAA and Fe-TIAA had higher SOD activity than other Fe and Cu complexes and protected Escherichiu coli cells from paraquat toxicity. In case of using tris[N-(Cmethyl-2-pyridylrnethyl)-2-aminoethyl]amine (6MeTPAA) as ligand, the Fe complex could not be obtained, which may be due to the steric hindrance of Cmethyl substituent. Generally, Cu complexes had low SOD activity, compared with Fe complexes, and could not suppress paraquat toxicity.     

Polymorphism of the SOD1-DNA aggregation species can be modulated by DNA

Proteinaceous aggregates rich in copper, zinc superoxide dismutase (SOD1) have been found in both in vivo and in vitro models. We have shown that double-stranded DNA that acts as a template accelerates the in vitro formation of wild-type SOD1 aggregates. Here, we examined the polymorphism of templated-SOD1 aggregates generated in vitro upon association with DNA under different conditions. Electron microscopy imaging indicates that this polymorphism is capable of being manipulated by the shapes, structures, and doses of the DNAs tested. The nanometer- and micrometer-scale aggregates formed under acidic conditions and under neutral conditions containing ascorbate fall into three classes: aggregate monomers, oligomeric aggregates, and macroaggregates. The aggregate monomers observed at given DNA doses exhibit a polymorphism that is markedly corresponded to the coiled shapes of linear DNA and structures of plasmid DNA. On the other hand, the regularly branched structures observed under both atomic force microscopy and optical microscope indicate that the DNAs tested are simultaneously condensed into a nanoparticle with a specific morphology during SOD1 aggregation, revealing that SOD1 aggregation and DNA condensation are two concurrent phenomena. The results might provide the basis of therapeutic approaches to suppress the formation of toxic protein oligomers or aggregates by screening the toxicity of the protein aggregates with various sizes and morphologies.     

Determination of superoxide dismutase-like activity in some divalent metal saccharinates

The superoxide-dismutase-like activity of a series of divalent metal saccharinates of general stoichiometry [M^II(Sac)₂(H₂O)₄]·2H₂O (with M^II=Mn,Fe,Co,Ni,Cu,Zn) has been investigated using the nitroblue tetrazolium O ₂ ⁻ reduction assay. The results show that all these complexes possess the capability to dismutate the superoxide anion generated in the xanthine/xanthine oxidase system. Interestingly, the greatest activity is shown by the corresponding copper complex. The results are discussed and compared with those obtained for native superoxide dismutase, which was tested under the same experimental conditions. Dedicated to Prof. Pedro J. Aymonino on the occasion of his 65^th birthday.     

Generation and characterization of cells that can be conditionally depleted of mitochondrial SOD2

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果     

Superoxide dismutase as a target enzyme for Fe-porphyrin-induced cell death

The cell viability of human cancer cell lines treated with [5,10-bis(N-methyl-4-pyridyl)-15,20-diphenyl]porphinatoiron(III) (cis-FeMPy(2)P(2)P) has been estimated. The cis-FeMPy(2)P(2)P is a superoxide dismutase (SOD) mimic in vitro that exhibited a significant toxicity in cancer cell lines. This toxicity is rather due to pro-oxidant properties of the iron-porphyrin in vivo. We have demonstrated that there was the relationship between the LD(50) values calculated from the viability of cancer cell lines treated with cis-FeMPy(2)P(2)P and the SOD activities of the cell lines. Furthermore, the inhibition of SOD by antisense S-oligonucleotide increased the cytotoxic effect of cis-FeMPy(2)P(2)P against cancer cells. These results suggest that SOD is a target enzyme for the cell death induced by cis-FeMPy(2)P(2)P as a new class of anticancer agents.     

Free radical scavenging activity of the Japanese herbal medicine Toki-Shakuyaku-San (TJ-23) and its effect on superoxide dismutase activity,lipid peroxides,glutamate, and monoamine metabolites in aged rat brain

The free radical scavenging activity of the Japanese herbal medicine, Toki-Shakuyaku-San (TJ-23; TSUMURA & Co., Tokyo, Japan), was examined using electron spin resonance (ESR) spectrometry. TJ-23 scavenged 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), superoxide (O₂ ⁻), and hydroxyl radicals (·OH) dose-dependently. It also diminished carbon centered radicals (·C) generated by oxidative stress and inhibited thiobarbituric acid-reactive substances (TBARS) formation in mouse cortex homogenate. In addition, the effect of TJ-23 on the concentration of neurotransmitters and TBARS formation, and superoxide dismutase (SOD) activity in the cortex, hippocampus and striatum of the aged rat brain was studied. The concentrations of the metabolites of monoamines, glutamate and glutamine were decreased by 4 weeks of oral administration of TJ-23. The SOD activity of mitochondrial fraction was increased and TBARS formation was significantly suppressed. These results suggest that TJ-23 has an antioxidant action and would have a prophylactic effect against free radical-mediated neurological diseases associated with aging.     

A model for gain of function in superoxide dismutase

Studies have found that mutant, misfolded superoxide dismutase [Cu–Zn] (SOD1) can convert wild type SOD1 (wtSOD1) in a prion-like fashion, and that misfolded wtSOD1 can be propagated by release and uptake of protein aggregates. In developing a prion-like mechanism for this propagation of SOD1 misfolding we have previously shown how enervation of the SOD1 electrostatic loop (ESL), caused by the formation of transient non-obligate SOD1 oligomers, can lead to an experimentally observed gain of interaction (GOI) that results in the formation of SOD1 amyloid-like filaments. It has also been shown that freedom of ESL motion is essential to catalytic function. This work investigates the possibility that restricting ESL mobility might not only compromise superoxide catalytic activity but also serve to promote the peroxidase activity of SOD1, thus implicating the formation of SOD1 oligomers in both protein misfolding and in protein oxidation.     

The induction of oxidative stress and cellular death by the drinking water disinfection by-products,dichloroacetate and trichloroacetate in J774.A1 cells

The in vitro toxicity of the drinking water disinfection by products dichloroacetate (DCA) and trichloroacetate (TCA) were studied using the J774A.1 macrophage cell line. DCA and TCA were added to cell cultures at concentrations ranging between 8-32 mM and incubated for 24, 36 and 60 h. DCA and TCA effects on cellular viability, lactate dehydrogenase (LDH) release and superoxide anion (SA) production by the cells, as well as superoxide dismutase (SOD) activities of the cells were determined. DCA and TCA caused time- and concentration-dependent increases in cellular death, in LDH release and production of SA by the cells. The compounds also caused modulations in SOD activities of the cells, with increases observed at the lower concentrations and/or shorter periods of incubations and suppression with the higher concentrations and/or longer periods of incubation. The results of the study indicate that DCA and TCA induce macrophage activation and that the activation is associated with cellular toxicity. Also, DCA and TCA are found to be equitoxic to J774.A1 cells.     

Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases

It has been shown that superoxide dismutase (SOD) can both potentiate and attenuate NO-mediated toxicity. This present study investigated the role of SOD and GSH in a sustained nitrosative and oxidative environment simulated by the nitric oxide (NO) and superoxide (O(2)(.-)) donor, 3-morpholinosydnonimine (SIN-1). We describe, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results show that SOD's effects on nitrosation are biphasic in nature i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD predominantly exhibits a pronitrosative effect, with a complete loss of antinitrosative effects noted at higher levels of GSH. Our findings likely reflect the complex and dynamic nature of SOD interactions with oxidative and nitrosative species.     

Cu,Zn superoxide dismutase increases intracellular calcium levels via a phospholipase C-protein kinase C pathway in SK-N-BE neuroblastoma cells

The superoxide dismutase isoenzymes (SOD) play a key role in scavenging, O*2- radicals. In contrast with previous studies, recent data have shown that human neuroblastoma cells are able to export the cytosolic Cu,Zn superoxide dismutase (SOD1), thus suggesting a paracrine role exerted by this enzyme in the nervous system. To evaluate whether SOD1 could activate intracellular signalling pathways, the functional interaction between SOD1 and human neuroblastoma SK-N-BE cells was investigated. By analyzing the surface binding of biotinylated SOD1 on SK-N-BE cells and by measuring intracellular calcium concentrations and PKC activity, we demonstrated that SOD1 specifically interacts in a dose-dependent manner with the cell surface membrane of SK-N-BE. This binding was able to activate a PLC-PKC-dependent pathway that increased intracellular calcium concentrations mainly deriving from the intracellular stores. Furthermore, we showed that this effect was independent of SOD1 dismutase activity and was totally inhibited by U73122, the PLC blocker. On the whole, these data indicate that SOD1 carries out a neuromodulatory role affecting calcium-dependent cellular functions.     

Development of novel small molecules for the treatment of ALS

Amyotrophic lateral sclerosis (ALS) is a rare and progressive neurodegenerative disease with unknown etiology. It is caused by the degeneration of motor neurons responsible for controlling voluntary muscles. It has been reported that mutations in the superoxide dismutase (SOD) 1 gene can lead to ALS. SOD1 abnormalities have been identified in both familial, as well as sporadic ALS cases. SOD2 is a highly inducible SOD that works in conjunction with SOD1. SOD2 can be induced through activation of NF-κBs. We previously reported that the novel small molecule, SRI-22818, increases NF-κB expression and activation and SOD2 levels in vitro and has activity in vivo in the SOD1-G93A reference model of ALS. We report herein the synthesis and biological evaluation of SRI-22818 analogs.     

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.     

Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series as tumour photosensitizers.

Although folate deficiency and increased requirements for folate are observed in most alcoholics, the possibility that acetaldehyde generated from ethanol metabolism may increase folate catabolism has not been previously demonstrated. Folate cleavage was studied in vitro during the metabolism of acetaldehyde by xanthine oxidase, measured as the production of p-aminobenzoylglutamate from folate using h.p.l.c. Acetaldehyde/xanthine oxidase generated superoxide, which cleaved folates (5-methyltetrahydrofolate greater than folinic acid greater than folate) and was inhibited by superoxide dismutase. Cleavage was increased by addition of ferritin and inhibited by desferrioxamine (a tight chelator of iron), suggesting the importance of catalytic iron. Superoxide generated from the metabolism of ethanol to acetaldehyde in the presence of xanthine oxidase in vivo may contribute to the severity of folate deficiency in the alcoholic.     

Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice

Mutations of cytosolic Cu/Zn superoxide dismutase 1 (SOD1) in humans and overexpression of mutant human SOD1 genes in transgenic mice are associated with the motor neuron degenerative condition known as amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease). Gain-of-function toxicity from the mutant protein expressed in motor neurons, associated with its misfolding and aggregation, leads to dysfunction and cell death, associated with paralyzing disease. Here, using hydrogen-deuterium exchange in intact mice in vivo, we have addressed whether an ALS-associated mutant protein, G85R SOD1-YFP, is subject to the same rate of turnover in spinal cord both early in the course of the disease and later. We find that the mutant protein turns over about 10-fold faster than a similarly expressed wild-type fusion and that there is no significant change in the rate of turnover as animals age and disease progresses.     

Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.     

SUMO-1 modification increases human SOD1 stability and aggregation

The mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause approximately 20% cases of familial amyotrophic lateral sclerosis (FALS), characterized by selective loss of motor neurons. Mutant SOD1 forms inclusions in tissues from FALS patients. However, the precise mechanism of the accumulation of mutant SOD1 remains unclear. Here we show that human SOD1 is a substrate modified by SUMO-1. A conversion of lysine 75 to an arginine within a SUMO consensus sequence in SOD1 completely abolishes SOD1 sumoylation. We further show that SUMO-1 modification, on both wild-type and mutant SOD1, increases SOD1 steady state level and aggregation. Moreover, SUMO-1 co-localizes onto the aggregates formed by SOD1. These findings imply that SUMO-1 modification on lysine 75 may participate in regulating SOD1 stability and its aggregation process. Thus, our results suggest that sumoylation of SOD1 may be involved in the pathogenesis of FALS associated with mutant SOD1.     

Hitchhiking of Cu/Zn Superoxide Dismutase to Peroxisomes – Evidence for a Natural Piggyback Import Mechanism in Mammals

Most newly synthesized peroxisomal proteins are imported in a receptor-mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so-called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner 'copper chaperone of SOD1' (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D-liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1-dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS-mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co-import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.