Erythrocyte carbonic anhydrase: a major intracellular enzyme to regulate cellular sodium metabolism in chronic renal failure patients with diabetes and hypertension.

Changes in carbonic anhydrase (CA) activity have been associated with metabolic diseases like diabetes mellitus and hypertension. To explore the exchange of H+ for Na+ and 22Na+, the sodium pool, CA activity and H2O content in erythrocytes from the two groups of diabetic chronic renal failure (CRF) patients with and without hypertension before dialysis were studied. The results were compared with those from the normotensive controls. The CA activity was determined spectrophotometrically, the sodium pool by ouabain insensitive 22Na+ influx and the percent H2O content gravimetrically. The 22Na+ influx in CRF patients with hypertension was significantly higher (p less than 0.025) than in the normotensive CRF patients and the controls. The levels of CA activity (U/min/mL) and the percent H2O content were significantly different in the hypertensive and the normotensive CRF patients from the control group (2.24 +/- 0.69 and 67.11 +/- 1.33, 1.95 +/- 0.63 and 66.43 +/- 1.51, 1.44 +/- 0.07 and 63.61 +/- 1.72, respectively). The present study implies a relationship between the 22Na+ influx and CA activity in CRF patients with hypertension. The variation of CA activity may thus result in changes in H+ production and ultimately in the intracellular Na+ pool.     

In vitro scavenger activity of some flavonoids and melanins against O2-(.).

The scavenger activity against O2-. of some flavonoids and melanins (synthetic melanins and melanins isolated from animal tissues, vegetable seeds, and mushroom spores) has been studied by ESR spectrometry. All these substances, except flavon and flavanone, diminish the signal of O2-. generated in vitro by a system containing H2O2 and acetone in an alkaline medium. It is shown that the presence of hydroxyl groups in the B ring of flavonoids is essential for their scavenger activity. Moreover, the presence of a hydroxyl at C-3 enhances the scavenger ability of flavonoids. Generally, aglycons are more active than their glycosides. It seems plausible that the antioxidant property of these substances comes from their scavenger activity against O2-(.). It is also pointed out that the scavenger activity shown by melanins, is strictly correlated with their nature of stable free radical.     

Natural selection and early changes of phenotype of tumor cells in vivo: acquisition of new defense mechanisms

This review summarizes results obtained in the author's and collaborating laboratories within the last decade and is designed to attract the attention of researchers to discrete biochemical mechanisms of protection acquired in vivo by cells of malignant tumors against effectors of innate antitumor immunity. Tumor progression in vivo is associated with the appearance and selection of tumor cells with new specific characteristics: a high level of H(2)O(2)-catabolizing (antioxidant) activity (H(2)O(2)CA) and the ability for immediate release of E2-type prostaglandin (PGES) on contact with natural killers, macrophages, and neutrophils; the expression of the [H(2)O(2)CA + PGES] phenotype provides the tumor cells with two mechanisms of local protection against effectors of innate and acquired antitumor immunity. This results in a 10-100-fold less effective rejection of tumor cells in immune and normal animals and corresponding increase of tumorigenicity. The in vitro transformation of normal fibroblasts, spontaneous or induced by oncogenes LTSV40, E1a,b, Ha-ras, myc, and also by p53(175) and bcl-2 does not result in the [H(2)O(2)CA + PGES] phenotype expression, but during subsequent in vivo growth of the above-mentioned transformants the selection of tumor cells of the [H(2)O(2)CA + PGES] phenotype is correlated with a 30-200-fold increase in their tumorigenicity (accompanied or not accompanied by spontaneous metastatic activity). Unlike the transformation induced by the above-mentioned oncogenes, the transformation of normal cells by the v-src gene results in the [H(2)O(2)CA + PGES] phenotype expression. The data presented confirm the determining role of the v-src gene in the expression of the [H(2)O(2)CA + PGES] phenotype. In various primary viral carcinogenesis (SV40, SA7(C8)) the natural selection of cells expressing the [H(2)O(2)CA + PGES] phenotype begins even within the latent period and can be completed by the appearance of primary tumors.     

Accumulation of antioxidant phenolic constituents in submerged cultures of Inonotus obliquus

Phenolic compounds produced by sclerotia of Inonotus obliquus are the active constituents responsible for antioxidant activities. In this study, I. obliquus was grown in a continuously stirred tank reactor (CSTR) to explore how it accumulates phenolic compounds in different culture media and whether these compounds possess antioxidant activities. Phenolic compounds produced by I. obliquus in the control medium consisted of melanins, flavonoids, polyphenols and small phenolics. Their accumulation was affected by adding H(2)O(2) to the medium, where increased levels of total intracellular phenols (TIP) and melanins, but less total extracellular phenol (TEP) occurred. Simultaneous exposure to H(2)O(2) and arbutin resulted in a further increase in TIP production and reduced accumulation of TEP. Both TIP and TEP obtained at different culture ages and media were active in scavenging superoxide anion and DPPH radicals. Therefore, production of phenolic compounds by I. obliquus is enhanced by imposing oxidative stress, which might allow it to be exploited as a reliable source of pharmaceutically important phenolic compounds.     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

Effects of Selenium on Calcium Signaling and Apoptosis in Rat Dorsal Root Ganglion Neurons Induced by Oxidative Stress

Ca(2+) is well known for its role as crucial second messenger in modulating many cellular physiological functions, Ca(2+) overload is detrimental to cellular function and may present as an important cause of cellular oxidative stress generation and apoptosis. The aim of this study is to investigate the effects of selenium on lipid peroxidation, reduced glutathione (GSH), glutathione peroxidase (GSH-Px), cytosolic Ca(2+) release, cell viability (MTT) and apoptosis values in dorsal root ganglion (DRG) sensory neurons of rats. DRG cells were divided into four groups namely control, H(2)O(2) (as a model substance used as a paradigm for oxidative stress), selenium, selenium + H(2)O(2). Moderate doses and times of H(2)O(2) and selenium were determined by MTT test. Cells were preterated 200 nM selenium for 30 h before incubatation with 1 μM H(2)O(2) for 2 h. Lipid peroxidation levels were lower in the control, selenium, selenium + H(2)O(2) groups than in the H(2)O(2) group. GSH-Px activities were higher in the selenium groups than in the H(2)O(2) group. GSH levels were higher in the control, selenium, selenium + H(2)O(2) groups than in the H(2)O(2) group. Cytosolic Ca(2+) release was higher in the H(2)O(2) group than in the control, selenium, selenium + H(2)O(2) groups. Cytosolic Ca(2+) release was lower in the selenium + H(2)O(2) group than in the H(2)O(2). In conclusion, the present study demonstrates that selenium induced protective effects on oxidative stress, [Ca(2+)](c) release and apoptosis in DRG cells. Since selenium deficiency is a common feature of oxidative stress-induced neurological diseases of sensory neurons, our findings are relevant to the etiology of pathology in oxidative stress-induced neurological diseases of the DRG neurons.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.     

The determination of the Mg2+.ATP dissociation constant by competition with Eu3+ ion using laser-induced Eu3+ ion luminescence spectroscopy

A direct spectroscopic method for the determination of the submicromolar dissociation constant of Eu3+. ATP using laser-induced Eu3+ ion luminescence spectroscopy is described. The dissociation constant of Mg2+.ATP is then determined by the competition of Mg2+ with Eu3+ for the binding of ATP. The experiments were performed in 2H2O to mitigate the significant quenching of the Eu3+ luminescence that occurs in 1H2O. Values for the effective dissociation constants of the 1:1 ATP metal ion complexes of 1.2 +/- 0.3 X 10(-7) and 2.7 +/- 0.7 X 10(-4) M are obtained for Eu3+ and Mg2+, respectively, at p2H 5.8.     

Iron oxidation chemistry in ferritin. Increasing Fe/O2 stoichiometry during core formation

The origin of previously observed variations in stoichiometry of iron oxidation during the oxidative deposition of iron in ferritin has been poorly understood. Knowledge of the stoichiometry of Fe(II) oxidation by O2 is essential to establishing the mechanism of iron core formation. In the present work, the amount of Fe(II) oxidized was measured by M?ssbauer spectrometry and the O2 consumed by mass spectrometry. The number of protons produced in the reaction was measured by "pH stat" titration and hydrogen peroxide production by the effect of the enzyme catalase on the measured stoichiometry. For protein samples containing low levels of iron (24 Fe(II)/protein) the stoichiometry was found to be 1.95 +/- 0.18 Fe(II)/O2 with H2O2 being a product, viz. Equation 1. 2Fe2+ + O2 + 4H2O----2FeOOH + H2O2 + 4H+ (1) EPR spin trapping experiments showed no evidence of superoxide radical formation. The stoichiometry markedly increased with additional iron (240-960 Fe/protein), to a value of 4 Fe(II)/O2 as in Equation 2. 4Fe2+ + O2 + 6H2O----4FeOOH + 8H+ (2) As the iron core is progressively laid down, the mechanism of iron oxidation changes from a protein dominated process with H2O2 being the primary product of O2 reduction to a mineral surface dominated process where H2O is the primary product. These results emphasize the importance of the apoferritin shell in facilitating iron oxidation in the early stage of iron deposition prior to significant development of the polynuclear iron core.     

Probing the geometric and electronic structures of the low-temperature azide adduct and the product-inhibited form of oxidized manganese superoxide dismutase

The geometric and electronic structures of the six-coordinate azide adduct of oxidized manganese superoxide dismutase (Mn3+ SOD) that is formed at low temperatures, LT N3-Mn3+ SOD, has been examined in detail through a combined spectroscopic/computational approach. Electronic absorption, circular dichroism (CD), magnetic CD (MCD) and variable-temperature, variable-field (VTVH) MCD spectroscopies were used to determine electronic transition energies and to obtain an estimate of zero-field splitting parameters for LT N3-Mn3+ SOD. These experimental data were utilized in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization-configuration interaction (INDO/S-CI) and time-dependent density functional theory (TD-DFT) computations to evaluate hypothetical active-site models of LT N3-Mn3+ SOD generated by constrained DFT geometry optimizations. Collectively, our spectroscopic/computational results indicate that N3- binding to Mn3+ SOD at low temperatures promotes neither protonation of the axial solvent ligand nor reorientation of the redox-active molecular orbital, both of which had been previously suggested. Using the same experimentally validated computational approach, models of the product-inhibited form of MnSOD were also developed and evaluated by their relative energies and TD-DFT-computed absorption spectra. On the basis of our computational results as well as previously published kinetic data, we propose that the product-inhibited form of MnSOD is best described as a side-on peroxo-Mn3+ adduct possessing an axial H2O ligand. Notably, attempts to generate a stable hydroperoxo-Mn3+ SOD species by protonation of the proximal O atom of the hydroperoxo ligand resulted in dissociation of HOO- and eventual H+ transfer from Tyr34 to HOO-, generating deprotonated Tyr34 and H2O2. The implications of these results with respect to the mechanism of O2*- dismutation by MnSOD are discussed.     

Peroxidase as an alternative to tyrosinase in the oxidative polymerization of 5,6-dihydroxyindoles to melanin(s).

The ability of the peroxidase/H2O2 system to promote the oxidative polymerization of 5,6-dihydroxyindole (DI) and 5,6-dihydroxyindole-2-carboxylic acid (DICA) to melanin pigments was investigated in comparison with tyrosinase. commonly regarded as the sole enzyme involved in melanogenesis. In 0.025 M phosphate buffer at pH 6.8, tyrosinase (2.7 x 10(-3) U/ml) induced a smooth oxidation of 3.0 x 10(-5) M DI (initial rate = 4.4 x 10(-5) M/s) to give a complex mixture of products with the 2,4'-dimer I as the main component, whereas, under the same conditions, peroxidase (0.44 U/ml) and 1.2 x 10(-4) M H2O2 caused the instantaneous conversion of the substrate to a well-defined pattern of products, comprising the 2,4'-and 2,7'-DI dimers I and II, and the related trimers III and IV. When 3.0 x 10(-5) M DICA was used as the substrate, the difference in the effectiveness of the enzymes was much more pronounced. Thus, while peroxidase accomplished the fast oxidation of the indole, yielding the dimer V and the trimer VI as the main products, tyrosinase proved unable to induce more than a poor and sluggish reaction with an initial rate of 5.6.10(-6) M/s. These results raise the possibility that peroxidase, rather than, or in addition to, tyrosinase, may play a critical role in the later stages of the biosynthesis of melanins.     

Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism

Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EA相似文献   

Ischemic preconditioning enhances scavenging activity of reactive oxygen species and diminishes transmural difference of infarct size

Reactive oxygen species (ROS) enhance myocardial ischemia-reperfusion (I/R) injury. Ischemic preconditioning (PC) provides potent cardioprotective effects in I/R. However, it has not been elucidated whether PC diminishes ROS stress in I/R and whether PC protects the myocardium from ROS stress transmurally and homogeneously. Isolated rabbit hearts perfused with Krebs-Henseleit buffer underwent 30 min of ischemia and 60 min of reperfusion. Hemodynamic changes and myocardial damage extent were analyzed in four groups. The control group underwent I/R alone. The H2O2 group underwent I/R with H2O2 infusion (50 microM) in the first minute of reperfusion to enhance oxidative stress. The PC and H2O2+PC groups underwent 5 min of PC before control and H2O2 protocols, respectively. Extracted myocardial DNA was analyzed for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, with the use of the HPLC-electrochemical detection method. Glutathione peroxidase (GPX) activity and the reduced form of GSH were measured by spectrophotometric assays. The myocardial infarct size was significantly reduced in the PC group (19 +/- 2%) compared with the control group (37 +/- 4%; P < 0.05), particularly in the subendocardium. H2O2 transmurally increased the infarct size by 59 +/- 4% (P < 0.05), which was significantly diminished in the H2O2+PC group (31 +/- 4%; P < 0.01). The GSH levels, but not GPX activity, were well preserved transmurally in protocols with PC. The 8-OHdG levels were significantly decreased in PC and were significantly enhanced in H2O2 (P < 0.01). These changes in oxidative DNA damage were effectively diminished by PC. In conclusion, PC enhanced the scavenging activity of GSH against ROS transmurally, reduced myocardial damage, particularly in the subendocardium, and diminished the transmural difference in myocardial infarct size.     

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.     

Sexual dimorphism in oxidant status in spontaneously hypertensive rats

Male spontaneously hypertensive rats (SHR) have a blunted pressure-natriuresis relationship and enhanced oxidative stress compared with female SHR. Furthermore, oxidative stress contributes to abnormal renal Na+ handling and renal damage in hypertension. The aim of this study was to determine whether a sex difference exists in renal inner medullary hydrogen peroxide (H2O2) levels and/or antioxidant systems in SHR and the influence of sex steroids on these systems. Thirteen-week-old intact and gonadectomized male and female SHR were placed in metabolic cages for 24-h urine collection. Renal inner medullas were isolated for antioxidant activity assays and Western blot analysis or for measurements of H2O2 using Amplex Red. Studies verified that male SHR had greater Na+ reabsorption compared with female SHR. Male SHR had enhanced urinary excretion of H2O2 compared with female SHR. Gonadectomy decreased H2O2 excretion in males and increased H2O2 excretion in females, suggesting that testosterone stimulates total body oxidative stress and estrogen suppresses levels of total body oxidative stress. There was not a sex difference in inner medullary H2O2 levels. Male SHR had a testosterone-dependent increase in inner medullary SOD activity, and both intact and gonadectomized males had high levels of inner medullary catalase activity compared with females. The results of this study showed that there was a sexual dimorphism in Na+ handling and oxidant status. We hypothesize that there is a testosterone-sensitive increase in whole body reactive oxygen species production that results in a compensatory increase in the inner medullary antioxidant capability possibly to normalize Na+ handling.     

Differential alterations in antioxidant capacity in cells from Alzheimer patients

Oxidative stress occurs in brains of Alzheimer's disease (AD) patients. A major question in AD research is whether the oxidative stress is just secondary to neurodegeneration. To test whether oxidative stress is an inherent property of AD tissues, the ability of cultured fibroblasts bearing the AD Presenilin-1 246 Ala-->Glu mutation to handle reactive oxygen species (ROS) was compared to controls. Although ROS in cells from AD subjects were only slightly less than cells from controls under basal conditions (-10%) or after exposure to H(2)O(2) (-16%), treatment with antioxidants revealed clear differences. Pretreatment with DMSO, a hydroxyl radical scavenger, reduced basal and H(2)O(2)-induced ROS levels significantly more in cells from controls (-22%, -22%) than in those from AD subjects (-4%, +14%). On the other hand, pretreatment with Trolox diminished H(2)O(2)-induced ROS significantly more in cells from AD (-60%) than control subjects (-39%). In summary, cells from AD patients have greater Trolox sensitive ROS and less DMSO sensitive ROS than controls. The results demonstrate that fibroblasts bearing this PS-1 mutation have altered means of handling oxidative stress and appear useful for determining the mechanism underlying the altered redox metabolism.     

Polyethylene glycol-attached antioxidant enzymes decrease pulmonary oxygen toxicity in rats

When exposed continuously to hyperoxia (100% O2, 760 Torr barometric pressure), rats pretreated with polyethylene glycol (PEG)-attached superoxide dismutase and catalase (PEG-SOD + PEG-CAT) lived longer (79.1 + 7.6 h) than rats pretreated with saline (60.7 +/- 2.1 h) or PEG-inactivated-SOD + PEG-inactivated-CAT (62.3 +/- 1.6 h). Rats pretreated with PEG-SOD + PEG-CAT also had less hyperoxia-induced acute oxidative edematous lung injury, as assessed by increases in lung oxidized glutathione (GSSG) contents, pleural effusions, and lung lavage albumin concentrations than saline-pretreated rats. Rats pretreated with the long-lived conjugates PEG-inactivated-SOD + PEG-inactivated-CAT or PEG-albumin also had decreased acute oxidative edematous lung injury compared with rats pretreated with PEG, SOD + CAT + PEG, SOD + CAT, or saline. In vitro studies suggested that PEG itself may have contributed to protection by scavenging hydroxyl radical (.OH) but not superoxide (O2-.) or H2O2. Compared with more effective endogenous (via preexposure to hypoxia) or exogenous (via liposomes) means for increasing lung antioxidant enzymes, PEG enzymes are less protective against lung injury from continuous hyperoxia.