Estrogen regulation of superoxide dismutase in normal rat mammary tissues and mammary tumors

Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors. The specific activities of $\text{Cu}\text{Zu}$ superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.     

Studies on the mechanism of toxicity of the mycotoxin, sporidesmin. I. Generation of superoxide radical by sporidesmin

Sporidesmin (SDMS2), the mycotoxin responsible for 'facial eczema' in ruminants, contains a disulphide group which appears to be intimately involved in its toxic action. The reduced (dithiol) form of sporidesmin has been shown readily to undergo autoxidation in vitro in a reaction which generates superoxide radical (O2-). The autoxidation reaction, which takes place over a wide pH range, is strongly catalysed by trace amounts of copper, although the reaction was inhibited at high concentrations of this metal. Inhibition of the autooxidation of reduced sporidesmin (SDM(SH)2) was also observed in the presence of nickel, cobalt and manganese. Superoxide radical is also generated from SDMS2 itself in a cyclic reduction/autoxidation reaction with glutathione and other thiols; in view of the known toxicity of superoxide and its derivatives, it is suggested that oxygen-free-radicals may be involved in the initiation of the deleterious effects of the mycotoxin.     

Promotion by phosphate of Fe(III)- and Cu(II)-catalyzed autoxidation of fructose

Although the oxidative destruction of glucose and fructose has been studied by several investigators over the past century, the mechanism by which phosphate promotes these oxidation reactions is not known. A wide range of oxidation products have been used to monitor the oxidation of sugars and free radicals have been shown to be involved. The influence of phosphate concentration on the rate of production of free radicals and several sugar oxidation products has been studied. It was found that fructose is much more susceptible to autoxidation than glucose, galactose, or sucrose. The promotion of sugar oxidation by phosphate was found to be iron dependent. Addition of the iron chelators, diethylenetriaminepentaacetic acid (DTPA) and desferrioxamine completely suppressed the oxidation reactions, even at high concentrations of phosphate. Formaldehyde was positively identified as a product of fructose oxidation by HPLC analysis of its acetylacetone adduct. A mechanism is proposed in which phosphate cleaves the oxo bridges of the iron(III)-fructose complex, based on UV spectral analysis and magnetic susceptibility measurements, and thereby catalyzes the autoxidation of fructose.     

High temperature limits in vivo pollen tube growth rates by altering diurnal carbohydrate balance in field-grown Gossypium hirsutum pistils

It has recently been reported that high temperature slows in vivo pollen tube growth rates in Gossypium hirsutum pistils under field conditions. Although numerous physical and biochemical pollen-pistil interactions are necessary for in vivo pollen tube growth to occur, studies investigating the influence of heat-induced changes in pistil biochemistry on in vivo pollen tube growth rates are lacking. We hypothesized that high temperature would alter diurnal pistil biochemistry and that pollen tube growth rates would be dependent upon the soluble carbohydrate content of the pistil during pollen tube growth. G. hirsutum seeds were sown on different dates to obtain flowers exposed to contrasting ambient temperatures but at the same developmental stage. Diurnal pistil measurements included carbohydrate balance, glutathione reductase (GR; EC 1.8.1.7), soluble protein, superoxide dismutase (SOD; EC 1.15.1.1), NADPH oxidase (NOX; EC 1.6.3.1), adenosine triphosphate (ATP), and water-soluble calcium. Soluble carbohydrate levels in cotton pistils were as much as 67.5% lower under high temperature conditions (34.6 °C maximum air temperature; August 4, 2009) than under cooler conditions (29.9 °C maximum air temperature; August 14, 2009). Regression analysis revealed that pollen tube growth rates were highly correlated with the soluble carbohydrate content of the pistil during pollen tube growth (r² = 0.932). Higher ambient temperature conditions on August 4 increased GR activity in the pistil only during periods not associated with in vivo pollen tube growth; pistil protein content declined earlier in the day under high temperatures; SOD and NOX were unaffected by either sample date or time of day; pistil ATP and water soluble calcium were unaffected by the warmer temperatures. We conclude that moderate heat stress significantly alters diurnal carbohydrate balance in the pistil and suggest that pollen tube growth rate through the style may be limited by soluble carbohydrate supply in the pistil.     

Role of Oxygen Free Radicals in Respiratory Distress Induced by Arachidonic Acid In the Rat

The purpose of our present study is the possible implication of oxygen free radicals in the respiratory distress induced in rats by intravenous administration of arachidonic acid (20mg/kg). The respiratory frequency was measured and plasma TXB2 concentration was assayed by RIA from blood withdrawn I min after arachidonic acid administration. The substances studied were: SOD, catalase, manifold, DMSO, BHT, imidazole. All the drugs, except imidazole, significantly protect the rats from the respiratory distress induced by arachidonic acid. SOD, catalase, BHT and imidatole inhibit whereas mannitol and DMSO increase the plasma levels of TXB2. We suggest that oxygen free radicals generated in the respiratory burst induced by arachidonic acid are mainly responsible for the consequent respiratory distress.     

Production of singlet oxygen and superoxide radicals by psoralens and their biological significance

We have investigated a series of linear and angular furocoumarins, capable of forming either the monofunctional adducts (single strand) or bifunctional adducts (interstrand cross-links) with DNA with a view to examine the relationship of their skin photosensitizing potency, their ability to produce singlet oxygen (1O2) or superoxide radicals (O-.2 or HO.2), and their carcinogenic activity. The significance of photochemical interactions of psoralens and DNA is well known in skin photosensitization and skin carcinogenesis. Our data suggest that both monofunctional and bifunctional psoralens produce 1O2 and O-.2, and these reactive forms of oxygen may contribute to the development of skin cancer and membrane-damaging effects of these furocoumarins.     

The coupling between disulphide status, metallation and dimer interface strength in Cu/Zn superoxide dismutase

The gain of neurotoxic function in amyotrophic lateral sclerosis (ALS) has been linked to misfolding of the homodimeric enzyme Cu/Zn superoxide dismutase (SOD). Here, we present the crystal structure of fully cysteine-depleted human SOD (SOD(CallA)), representing a reduced, marginally stable intermediate on the folding pathway in vivo that has also been implicated as neurotoxic precursor state. A hallmark of this species is that it fails to dimerize and becomes trapped as a monomer in the absence of the active-site metals. The crystallographic data show that removal of the C57-C146 disulphide bond sets free the interface loop IV in the apo protein, whereas the same loop remains unaffected in the holo protein. Thus, the low dimerisation propensity of disulphide-reduced apoSOD seems to be of entropic origin due to increased loop flexibility in the monomeric state: in the disulphide-reduced holo protein this gain in configurational entropy upon splitting of the dimer interface is reduced by the metal coordination.     

Oxygen radical formation induced by gossypol in rat liver microsomes and human sperm

Gossypol, a polyphenolic compound found in cotton plants, has many potential uses, including use as a male antifertility drug and spermicide. Gossypol affects a variety of cell processes and many of these effects may be explained by a common underlying mechanism. Here we report that gossypol promotes the formation of oxygen radicals when incubated with rat liver microsomes and human sperm suggesting that oxygen radical production may be the underlying basis of its biological activity.     

Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice

Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogen’s effect in the brain, we administered hydrogen-rich pure water (H₂) to senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), also a well-known anti-oxidant. These KO mice were divided into three groups; recipients of H₂, VC, or pure water (H₂O), administered for 33 days. VC levels in H₂ and H₂O groups were <6% of those in the VC group. Subsequently, superoxide formation during hypoxia-reoxygenation treatment of brain slices from these groups was estimated by a real-time biography imaging system, which models living brain tissues, with Lucigenin used as chemiluminescence probe for superoxide. A significant 27.2% less superoxide formed in the H₂ group subjected to ischemia-reperfusion than in the H₂O group. Thus hydrogen-rich pure water acts as an anti-oxidant in the brain slices and prevents superoxide formation.     

Hydrogen peroxide oxidation by catalase-peroxidase follows a non-scrambling mechanism

Despite catalyzing the same reaction (2 H2O2-->2 H2O+O2) heme-containing monofunctional catalases and bifunctional catalase-peroxidases (KatGs) do not share sequence or structural similarities raising the question of whether or not the reaction pathways are similar or different. The production of dioxygen from hydrogen peroxide by monofunctional catalases has been shown to be a two-step process involving the redox intermediate compound I which oxidizes H2O2 directly to O2. In order to investigate the origin of O2 released in KatG mediated H2O2 degradation we performed a gas chromatography-mass spectrometry investigation of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing KatGs from Mycobacterium tuberculosis and Synechocystis PCC 6803. The GC-MS analysis clearly demonstrated the formation of (18)O2 (m/e = 36) and (16)O2 (m/e = 32) but not (16)O(18)O (m/e = 34) in the pH range 5.6-8.5 implying that O2 is formed by two-electron oxidation without breaking the O-O bond. Also active site variants of Synechocystis KatG with very low catalase but normal or even enhanced peroxidase activity (D152S, H123E, W122F, Y249F and R439A) are shown to oxidize H2O2 by a non-scrambling mechanism. The results are discussed with respect to the catalatic mechanism of KatG.     

Interaction between copper and zinc in metal accumulation in rats with particular reference to the synthesis of induced-metallothionein

The effectiveness of Zn at moderating the pro-oxidant effects of Cu was evaluated in two rat models that differed in the route and mode of administration. The endpoints investigated included measurement of the concentrations of Cu, Zn, metallothionein and glutathione concentrations, as well as SOD and catalase activity, in liver, kidneys and intestine. In a sub-chronic animal model, the hepatic accumulation of Cu was achieved by administration of dietary Cu (1.8 g/kg solid diet) for 30 days after which oral Zn (6g/kg solid diet) was given. Cu treatment induced an increase in the hepatic and intestinal concentration of Cu of 66 and 455%, respectively, that was not associated with synthesis of metallothionein synthesis, but rather appeared to be related to the higher activity of SOD. Subsequent administration with Zn after dietary Cu induced an increase in the hepatic and intestinal metallothionein content of more twice and reduced the Cu content to control values. Thus, Zn could act as both a competitor for absorption on the luminal side of the intestinal epithelium inducing the synthesis of metallothionein. In the second animal model, we studied the effects of interaction between Cu and Zn administered by i.p. injection at the dose of 3 and 10mg/kg, respectively; Zn was administered subsequent to Cu overload. In this case, when Zn was administered, Cu was already deposited in tissues and thus there is no competition between two metals at the level of membrane transport. In this experimental model treatment with Cu alone induced liver metallothionein synthesis, and the subsequent treatment with Zn did not decrease the hepatic content of Cu. One explanation for these observations is that Zn induces the synthesis of metallothionein, which binds Cu for which it has a higher affinity. Moreover, after treatment with Zn, SOD activity in the liver decreases of almost 30% with respect to treatment with alone Cu, suggesting that Zn has a protective effect.     

Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase

The effects of exogenous silicon (Si) on plant growth, activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase, and concentrations of ascorbate and glutathione were investigated in cucumber (Cucumis sativus L.) plants treated with excess manganese (Mn) (600 microM). Compared with the treatment of normal Mn (10 microM), excess Mn significantly increased H2O2 concentration and lipid peroxidation indicated by accumulation of thiobarbituric acid reactive substances. The leaves showed apparent symptoms of Mn toxicity and the plant growth was significantly inhibited by excess Mn. The addition of Si significantly decreased lipid peroxidation caused by excess Mn, inhibited the appearance of Mn toxicity symptoms, and improved plant growth. This alleviation of Mn toxicity by Si was related to a significant increase in the activities of SOD, APX, DHAR and GR and the concentrations of ascorbate and glutathione.     

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Growth promotion and a decrease of oxidative stress in maize seedlings by a combination of geomagnetic and weak electromagnetic fields

In the present study, we hypothesized that an appropriate combination of a geomagnetic field (as a static field) and an alternative magnetic field may result in the promotion of maize seedling growth by an alleviation of an excess production of reactive oxygen species. First, we determined the applicable range of frequencies by theoretical calculations, and a combined magnetic field was designed. The seeds were germinated in the magnetic field for 4 days, and the seedlings were allowed to grow in a nutrient solution for another 4 days. The magnetic field-treated maize seeds produced seedlings with a faster growth rate than the control seeds. The activity of superoxide dismutase in the magnetic field-treated seedlings was lower, while the total antioxidant capacity of these seedlings was higher than that of the control group. The maintenance of membrane integrity and a decrease of iron content in the magnetic field-treated seedlings suggest that a combination of both static and alternative magnetic fields promotes the growth of the plants by lowering iron absorption, a reduction in the Fenton chemistry, and lowering the risk of oxidative burst.     

The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation.These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor.     

The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals () generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1-10 μM) resulted in decreased formation of EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions.     

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant ‘Quickstart II’ and salt-sensitive ‘DP1′ were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4 d after treatment (DAT), but a lower level of enzyme activity at 8 and 12 d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.     

Tipburn in salt-affected lettuce (Lactuca sativa L.) plants results from local oxidative stress

Tipburn in lettuce is a physiological disorder expressed as a necrosis in the margins of young developing leaves and is commonly observed under saline conditions. Tipburn is usually attributed to Ca²⁺ deficiencies, and there has very limited research on other mechanisms that may contribute to tipburn development. This work examines whether symptoms are mediated by increased reactive oxygen species (ROS) production.Two butter lettuce (Lactuca sativa L.) varieties, Sunstar (Su) and Pontina (Po), with contrasting tipburn susceptibility were grown in hydroponics with low Ca²⁺ (0.5 mM), and with or without 50 mM NaCl. Tipburn symptoms were observed only in Su, and only in the saline treatment. Tipburn incidence in response to topical treatments with Ca²⁺ scavengers, Ca²⁺ transport inhibitors, and antioxidants was assessed. All treatments were applied before symptom expression, and evaluated later, when symptoms were expected to occur. Superoxide presence in tissues was determined with nitro blue tetrazolium (NBT) and oxidative damage as malondialdehyde (MDA) content. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities were assayed.Under control and saline conditions, tipburn could be induced in both varieties by topical treatments with a Ca²⁺ scavenger (EGTA) and Ca²⁺ transport inhibitors (verapamil, LaCl₃) and reduced by supplying Ca²⁺ along with a ionophore (A 23187). Tipburn symptoms were associated with locally produced ROS. O₂⁻ and oxidative damage significantly increased in leaf margins before symptom expression, while topical antioxidant applications (Tiron, DPI) reduced symptoms in treated leaves, but not in the rest of the plant. Antioxidant enzyme activity was higher in Po, and increased more in response to EGTA treatments, and may contribute to mitigating oxidative damage and tipburn expression in this variety.     

Effect of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine on calcium fluxes by human platelet microsomes

Under conditions where optimal concentrations of arachidonic acid, phosphatidic acid, or the calcium ionophore A23187 caused release of 50-95% of calcium from preloaded platelet microsomes, basophil platelet activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) did not cause the release of calcium at concentrations as high as 2 X 10(-5) M. The failure to stimulate calcium release was not due to metabolism or inactivation of AGEPC. These results show that AGEPC is not a calcium ionophore and is unable to directly effect the release of calcium from microsomes by mechanisms other than ionophoric action. The increase in intracellular levels that occurs during AGEPC-induced platelet aggregation must be an indirect effect of the AGEPC.