Oxidative stress in Caenorhabditis elegans: protective effects of superoxide dismutase/catalase mimetics

The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed.     

New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins

Mimetics of antioxidant enzymes such as superoxide dismutases (SOD) or catalases are reported as potential new drugs able to reduce oxidative stress damage. In particular, manganese(III) complexes of salen-type ligands have been studied as both SOD and catalase mimetics. In this paper, we report the synthesis of two novel conjugates of salen-type ligands with the β-cyclodextrin, the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N′-bis(salicylidene))]-β-cyclodextrin and the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N′-bis(3-methoxysalicylidene))]-β-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method. The catalase like activity was tested using a Clark-type oxygen electrode. The peroxidase activity was tested using the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The glycoconjugation of salen-manganese(III) complexes yields compounds with enhanced SOD activity. These complexes also show catalase and peroxidase activities higher than the simple salen complexes (EUK 113 and EUK 108).     

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol.

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress.     

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.     

No increase in lifespan in Caenorhabditis elegans upon treatment with the superoxide dismutase mimetic EUK-8

The superoxide dismutase mimetic EUK-8 has been reported to extend lifespan in the nematode Caenorhabditis elegans. However, in five trials administering EUK-8 in liquid culture with E. coli, and two trials using defined liquid medium, we observed no increase in C. elegans lifespan. Instead we saw a dose-dependent reduction of lifespan and fertility. We conclude that extension of C. elegans lifespan by EUK-8 may only occur under very particular culture conditions.     

Dual effects of copper-zinc superoxide dismutase

Copper-zinc superoxide dismutase (CuZnSOD) specifically catalyzes the removal of superoxide radicals to protect cellular function against the generation of superoxide-dependent hydroxyl radicals ((.)OH). However, an unexpected observation reveals that denatured CuZnSOD (dCuZnSOD) itself induces (.)OH formation. This dCuZnSOD-dependent (.)OH generation was not inhibited by active CuZnSOD, suggesting that it is a superoxide-independent process. Sodium cyanide, histidine, and N,N'-diethyldithiocarbamate abolished (.)OH generation, implying that Cu may be responsible for dCuZnSOD-induced (.)OH formation. Catalase eliminated ()OH generation, suggesting that hydrogen peroxide may be involved in the mechanism of dCuZnSOD-mediated (.)OH production. Furthermore, nitric oxide ((.)NO) completely inhibited dCuZnSOD-induced (.)OH radical generation, indicating that (.)NO is an important (.)OH radical scavenger. Our results shed new light on the effect of dysfunctional CuZnSOD and suggest that structural disorder of the enzyme may be one of the endogenous pathways of toxic (.)OH formation in biological systems.     

Prooxidant activity of the superoxide dismutase (SOD)-mimetic EUK-8 in proliferating and growth-arrested Escherichia coli cells

Numerous studies have aimed to alleviate oxidative stress in a wide range of organisms by increasing superoxide dismutase (SOD) activity. However, experimental approaches have yielded contradictory evidence, and kinetics models have shown that increases in SOD activity may increase, decrease, or not change hydrogen peroxide (H2O2) production, depending on the balance of the various processes that produce and consume superoxide (O2-). In this study we tested whether administration of EUK-8, a synthetic mimetic of the SOD enzyme, can protect starving Escherichia coli cells against stasis-induced oxidative stress. Surprisingly, administration of EUK-8 to starving E. coli cells enhances the production of reactive oxygen species (ROS), resulting in a massive increase of oxidative damage and replicative death of the bacteria. Our results confirm that manipulation of ROS levels by increasing SOD activity does not necessarily result in a consequent decline of oxidative stress and can yield opposite results in a relatively simple model system such as starving E. coli cells.     

Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans

According to the oxidative damage theory a primary cause of aging is the accrual of molecular damage from reactive oxygen species (ROS), particularly superoxide and its derivatives. This predicts that treatments that reduce ROS levels should retard aging. Using the nematode Caenorhabditis elegans, we tested the effects on stress resistance and life span of treatment with EUK-8 and EUK-134, synthetic mimetics of the antioxidant enzyme superoxide dismutase (SOD), which neutralises superoxide. Treatment with SOD mimetics elevated in vivo SOD activity levels, particularly in mitochondria, where up to 5-fold increases in SOD activity were recorded. Treatment with exogenous SOD mimetics did not affect endogenous protein SOD levels. Where life span was reduced by the superoxide generators paraquat and plumbagin, EUK-8 treatment increased life span in a dose-dependent fashion. Yet in the absence of a superoxide generator, treatment with EUK-8 or EUK-134 did not increase life span, even at doses that were optimal for protection against pro-oxidants. Thus, an elevation of SOD activity levels sufficient to increase life span when it is limited by superoxide generators does not retard aging in the absence of superoxide generators. This suggests that C. elegans life span is not normally limited by levels of superoxide and its derivatives.     

Mitigation of cocaine-mediated mitochondrial damage,defective mitophagy and microglial activation by superoxide dismutase mimetics

ABSTRACT

Although cocaine exposure has been shown to potentiate neuroinflammation by upregulating glial activation in the brain, the role of mitophagy in this process remains an enigma. In the present study, we sought to examine the role of impaired mitophagy in cocaine-mediated activation of microglia and to determine the ameliorative potential of superoxide dismutase mimetics in this context. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to cocaine resulted in decreased mitochondrial membrane potential, that was accompanied by increased expression of mitophagy markers, PINK1 and PRKN. Exposure of microglia to cocaine also resulted in increased expression of DNM1L and OPTN with a concomitant decrease in the rate of mitochondrial oxygen consumption as well as impaired mitochondrial functioning. Additionally, in the presence of cocaine, microglia also exhibited upregulated expression of autophagosome markers, BECN1, MAP1LC3B-II, and SQSTM1. Taken together, these findings suggested diminished mitophagy flux and accumulation of mitophagosomes in the presence of cocaine. These findings were further confirmed by imaging techniques such as transmission electron microscopy and confocal microscopy. Cocaine-mediated activation of microglia was further monitored by assessing the expression of the microglial marker (ITGAM) and the inflammatory cytokine (Tnf, Il1b, and Il6) mRNAs. Pharmacological, as well as gene-silencing approaches aimed at blocking both the autophagy/mitophagy and SIGMAR1 expression, underscored the role of impaired mitophagy in cocaine-mediated activation of microglia. Furthermore, superoxide dismutase mimetics such as TEMPOL and MitoTEMPO were shown to alleviate cocaine-mediated impaired mitophagy as well as microglial activation.

Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A₁; BECN1: beclin 1, autophagy related; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-Diamidino-2-phenylindole; DRD2: dopamine receptor D2; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; NTRK2: neurotrophic receptor tyrosine kinase 2; OCR: oxygen consumption rate; OPTN: optineurin; PBS: phosphate buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor     

Comparison of the effects of superoxide dismutase and cytochrome c on luminol chemiluminescence produced by xanthine oxidase-catalyzed reactions

Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.     

Dual effects of superoxide dismutase on the autoxidation of 1,4-naphthohydroquinone

The autoxidation of 1,4-naphthohydroquinone, in a phosphate, EDTA buffer at pH 7.4, exhibits an autocatalysis whose lag phase becomes more pronounced in the presence of either the Cu,Zn- or the Mn-containing superoxide dismutases. In contrast, the autoxidation of a second aliquot of the hydroquinone, added after complete oxidation of the first, is linear and is accelerated by superoxide dismutase. Catalase or inactive superoxide dismutase were without effect in either situation. These results are explicable in terms of a free radical chain reaction which is initially propagated by O2- and then, as the quinone accumulates, by univalent reduction of the quinone by the hydroquinone. Reduction of the quinone by O2- diminishes the overall rate of oxidation. It is not necessary to postulate catalysis by superoxide dismutase of the reduction of the semiquinone by O2-.     

Oxygen effects on maize leaf superoxide dismutase and glutathione reductase

Exposure of maize seedlings to an atmosphere containing 75% O₂ and 350 ppm CO₂ resulted in a two- to three-fold increase in glutathione reductase activity in leaf tissue within 48 hr after initiation of the O₂ treatment. This elevated level of glutathione reductase was still evident in plants maintained in the hyperopic environment for 7 days. Superoxide dismutase activity was not altered from its level in control tissue during the 7-day experimental period. These results suggest a key role for glutathione reductase in the protection of photosynthetic tissue against detrimental effects of intermediate reduction products of O₂.     

Effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the housefly,Musca domestica

The purpose of this study was to determine whether superoxide dismutase/catalase mimetics lengthen the life span of the housefly, Musca domestica, as previously demonstrated for the nematode Caenorhabditis elegans. Various concentrations of Eukarion-8 or Eukarion-134 were administered via the drinking water and the effects on the life span of the flies and amounts of protein carbonyls were determined under normoxic and hyperoxic conditions. These SOD/catalase mimetics neither extended the life span of the flies nor attenuated the protein carbonyl content under normoxic conditions and shortened life span under hyperoxic conditions. Thus, the effect of these SOD/catalase mimetics on the life span of animals seem to be species-specific.     

The effects of pH and temperature on the assay of superoxide dismutase.

A simple and reliable method for the measurement of superoxide dismutase (EC 1.15.1.1) activity is described. The method is based on a linear inhibition of the reduction of acetylated cytochrome c by superoxide dismutase.     

Inhibition of vancomycin-induced nephrotoxicity by targeting superoxide dismutase to renal proximal tubule cells in the rat

Vancomycin, a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). Because vancomycin induces renal dysfunction, the dose and the duration of its administration are limited. The mechanism of vancomycin-induced renal dysfunction is not known. We recently synthesized a hexamethylenediamine-conjugated cationic superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. The present work reports the protective effects of AH-SOD against vancomycin-induced renal dysfunction. Male Wistar rats (200-210 g) were intraperitoneally administered with either 200 or 400 mg/kg of vancomycin twice a day for 7 days. Either 5 mg/kg/day AH-SOD or saline was subcutaneously injected 5 min before every vancomycin injection. Biochemical analysis revealed that plasma levels of blood urea nitrogen and creatinine increased significantly in vancomycin-treated group by an AH-SOD-inhibitable mechanism. Histological examination revealed that vancomycin also elicited a marked destruction of glomeruli and necrosis of proximal tubule by an AH-SOD inhibitable mechanism. These results suggest that oxidative stress underlies the pathogenesis of vancomycin-induced nephrotoxicity and that targeting SOD and/or related antioxidants to renal proximal tubule cells might permit the administration of higher doses of vancomycin sufficient for eradication of MRSA without causing renal injury.     

The effects of oxygen on alkaline solutions of superoxide dismutase.

The blowing of oxygen through strongly alkaline solutions of SOD leads to the drop of pH by more than 3 units. The rate of the process depends linearly on the concentration of SOD. The effect of oxygen on the modification of the shape and the decrease of the intensity of EPR signal of SOD were observed. The incubation of strongly alkaline solutions of SOD under vacuum leads to the reduction of the protein copper. The data obtained suggest, that the reduced copper may be at least partially reoxidized by oxygen. It is suggested that at pH 12.5 and higher in the presence of SOD the reaction of electron transfer from hydroxyl anion to the oxygen takes place: $\text{OH}{}^{\mathrm{?}}\text{+ O}{}_2\text{→ OH + O}{}^{\mathrm{?}}{}_{\mathrm{<mtext>2</mtext><mtext>?</mtext>}}$     

Scavenging ROS: superoxide dismutase/catalase mimetics by the use of an oxidation-sensitive nanocarrier/enzyme conjugate

Reactive Oxygen Species (ROS) are quintessential inflammatory compounds with oxidizing behavior. We have successfully developed a micellar system with responsiveness at the same time to two of the most important ROS: superoxide and hydrogen peroxide. This allows for an effective and selective capture of the two compounds and, in perspective, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity toward superoxide with that of hydrophobic thioethers toward hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then "mopped-up" by the polysulfides: this modus operandi is functionally analogous to the SOD/catalase combination, with the advantages of (a) being based on a single and more stable system, and (b) a higher overall efficiency due the physical proximity of the two ROS-reactive centers (SOD and polysulfides).     

Phenotypes of mice lacking extracellular superoxide dismutase and copper- and zinc-containing superoxide dismutase

Mice lacking the secreted extracellular superoxide dismutase (EC-SOD) or the cytosolic copper- and zinc-containing SOD (CuZn-SOD) show relatively mild phenotypes. To explore the possibility that the isoenzymes have partly overlapping functions, single and double knockout mice were examined. The absence of EC-SOD was found to be without effect on the lifespan of mice, and the reduced lifespan of CuZn-SOD knockouts was not further shortened by EC-SOD deficiency. The urinary excretion of isoprostanes was increased in CuZn-SOD knockout mice, and plasma thiobarbituric acid-reactive substances levels were elevated in EC-SOD knockout mice. These oxidant stress markers showed potentiated increases in the absence of both isoenzymes. Other alterations were mainly found in CuZn-SOD knockout mice, such as halved glutathione peroxidase activity in the tissues examined and increased glutathione and iron in the liver. There were no changes in tissue content of the alternative superoxide scavenger ascorbate, but there was a 25% reduction in ascorbate in blood plasma in mice lacking CuZn-SOD. No increase was found in the urinary excretion of the terminal metabolites of NO, nitrite, and nitrate in any of the genotypes. In conclusion, apart from the increases in the global urinary and plasma oxidant stress markers, our phenotype studies revealed no other evidence that the copper- and zinc-containing SOD isoenzymes have overlapping roles.     

The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats

Thirty-four day old, ovariectomised rats were treated with increasing doses of estradiol, 2-hydroxyestradiol 2,3-dimethyl ether (23E₂), 4-hydroxyestradiol 3,4-dimethyl ether (34E₂) and 4-methoxy-estradiol (4ME₂) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E₂ and 34E₂ treatment reduced the enzyme activity significantly. Though 4ME₂ showed a decrease in NADH oxidase at 0.05μg/100gm body weight there was no further decrease at higher dose (5μg/100gm). The Superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while 23E₂, 34E₂, and 4ME₂ significantly reduced SOD in both liver and uterus. These results indicate that 23E₂, 34E₂ and 4ME₂, in spite of their non-uterotrophic property, affect uterine metabolism. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogens are observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogens may play an important role in the pathophysiology of certain tumors.