Vasoactive intestinal peptide, a singlet oxygen quencher

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.     

Insulin-like growth factor I receptor is expressed at normal levels in Nijmegen breakage syndrome cells

Curcumin (diferuloylmethane) is a major component of food flavoring turmeric (Curcuma longa), and has been reported to be anticarcinogenic and anti-inflammatory. Although curcumin was shown to have antioxidant properties, its exact antioxidant nature has not been fully investigated. In this report we have investigated the possible antioxidant properties of curcumin using EPR spectroscopic techniques. Curcumin was found to inhibit the (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose-dependent manner. (1)O(2) was produced in a photosensitizing system using rose bengal as sensitizer, and was detected as TEMP-(1)O(2) adducts by electron paramagnetic resonance (EPR) spectroscopic techniques using TEMP as a spin-trap. Curcumin at 2.75 microM caused 50% inhibition of TEMP-(1)O(2) adduct formation. However, curcumin only marginally inhibited (24% maximum at 80 microM) reduction of ferricytochrome c in a xanthine-xanthine oxidase system demonstrating that it is not an effective superoxide radical scavenger. Additionally, there was minor inhibition of DMPO-OH adduct formation by curcumin (solubilized in ethanol) when an ethanol control was included in the EPR spin-trapping study, suggesting that curcumin may not be an effective hydroxyl radical scavenger. Together these data demonstrate that curcumin is able only to effectively quench singlet oxygen at very low concentration in aqueous systems.     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.     

Manganese complexes of curcumin analogues: evaluation of hydroxyl radical scavenging ability, superoxide dismutase activity and stability towards hydrolysis

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO*) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 microM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2- where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2- to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO* scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+ releasing, respectively.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide

Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.     

Melatonin counteracts potentiation by homocysteine of KCL-induced vasoconstriction in human umbilical artery: relation to calcium influx

Homocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (HCY)-induced vascular disease. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of HCY on the human umbilical artery. Helical umbilical arterial strips without endothelium were obtained at elective Cesarean delivery near term. Changes in potassium chloride (KCl)-induced vasoconstriction were measured. Arterial strips were treated with HCY (10 or 100 microM) plus FeSO(4) (10 microM) alone or pretreated with a hydroxyl radical ((*)OH) scavenger, mannitol (20 mM), or MLT (1 or 10 microM). The effect of HCY on the response of arterial strips to external calcium (Ca(2+)) in the presence of KCl (20 mM) was determined. HCY plus FeSO(4) potentiated KCl-induced vasoconstriction in a concentration-dependent manner; pretreatment with mannitol significantly reduced this vasospastic effect. HCY (100 microM) significantly augmented the contractile response to external Ca(2+). MLT (10 microM) significantly suppressed the contractile response to external Ca(2+). These results suggest that HCY potentiates KCl-induced umbilical artery vasoconstriction, in part by increasing Ca(2+) influx in vascular smooth muscle cells via activation of Ca(2+) channels. MLT significantly suppressed the vasoconstrictive effect of HCY, probably by scavenging (*)OH arising from HCY autooxidation.     

Spinal cord injury increases iron levels: catalytic production of hydroxyl radicals

This study used a weight drop impact injury model to explore the role of iron and the reality of iron-catalyzed hydroxyl radical ((*)OH) formation in secondary spinal cord injury (SCI). The time course of total extracellular iron was measured following SCI by microcannula sampling and atomic absorption spectrophotometry analysis. Immediately following SCI, the total iron concentration increased from an undetectable level to an average of 1.32 microM. The time course of SCI-induced (*)OH-generating catalytic activity in the cord was obtained by determining the ability of tissue homogenate to convert hydrogen peroxide to (*)OH and then measuring 2,3-dihydroxybenzoic acid, a hydroxylation product of salicylate. The concentration of 2,3-DHBA quickly and significantly increased (p <.001) and returned to sham level (p = 1) by 30 min post-SCI. Desferrioxamine (80 and 800 mg/kg body weight) significantly (p <.001) reduced the catalytic activity, suggesting that iron is the major contributor of the activity. Administering FeCl(3) (100 microM)/EDTA (0.5 mM) in ACSF into the cord through a dialysis fiber significantly increased SCI-induced (*)OH production in the extracellular space, demonstrating that Fe(3+) can catalyze (*)OH production in vivo. Our results support that iron-catalyzed (*)OH formation plays a role in the early stage of secondary SCI.     

Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid, formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure.     

Protective effect of melatonin against homocysteine-induced vasoconstriction of human umbilical artery

Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of Hcy on the human umbilical artery. In an initial experiment in a cell-free system, a micromolar concentration of iron was found to catalyze oxygen-dependent oxidation of Hcy. MLT (10 or 100 microM) did not affect oxygen-dependent oxidation of Hcy. Next, smooth muscle contraction induced by prostaglandin F(2alpha) (10 microM) was measured in arterial strips. Hcy (10 to 500 microM) increased this vascular tension in a concentration-dependent manner (P < 0.0001). Addition of Fe(2+) (10 microM) significantly potentiated the Hcy effect. Removal of endothelium (P < 0.05), pretreatment with a nitric oxide (NO) synthesis inhibitor (l-N(G)-monomethylarginine, 200 microM, P < 0.001), or pretreatment with a hydroxyl radical ((*)OH) scavenger (mannitol, 10 mM, P < 0.001) significantly attenuated contraction potentiated by Hcy plus Fe(2+). At a much lower concentration than mannitol, MLT (1 to 100 microM) significantly reduced the contractile effect of Hcy and Fe(2+) in a concentration-dependent manner. Hcy plus Fe(2+) significantly impaired calcium ionophore A 23187-induced relaxation (P < 0.0001), while MLT restored this relaxation in a concentration-dependent manner. These findings suggest that Hcy potentiates vascular tension in human umbilical artery, possibly by suppressing bioavailable NO. MLT protects against the vasoconstrictive effect of Hcy, most likely by scavenging (*)OH arising from Hcy autooxidation.     

Free radical scavenging and antioxidant effects of lactate ion: an in vitro study.

Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.     

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.     

Soluble amyloid beta1-28-copper(I)/copper(II)/Iron(II) complexes are potent antioxidants in cell-free systems

Amyloid beta (Abeta) is a central characteristic of Alzheimer's disease (AD). Currently, there is a long-standing dispute regarding the role of Abeta-metal ion (Zn, Cu, and Fe) complexes in AD pathogenesis. Here, we aim to decipher the connection between oxidative damage implicated in AD and Abeta-metal ion complexes. For this purpose we study, using ESR, the modulation of Cu/Fe-induced H 2O 2 decomposition by Abeta 1-28 (Abeta 28), a soluble model of Abeta 40/42. The addition of H 2O 2 to 0.6 nM-360 microM Abeta 28 solutions containing 100 microM Cu(II)/Cu(I)/Fe(II) at pH 6.6 results in a concentration-dependent sigmoidal decay of [*OH] with IC 50 values of 61, 59, and 84 microM, respectively. Furthermore, Abeta 28 reduces 90% of *OH production rate in the Cu(I)-H 2O 2 system in 5 min. Unlike soluble Abeta 28, Abeta 28-Cu aggregates exhibit poor antioxidant activity. The mode of antioxidant activity of soluble Abeta 28 is twofold. The primary (rapid) mechanism involves metal chelation, whereas the secondary (slow) mechanism involves (*)OH scavenging and oxidation of Cu(Fe)-coordinating ligands. On the basis of our findings, we propose that soluble Abeta may play a protective role in the early stages of AD, but not in healthy individuals, where Abeta's concentration is nanomolar. Yet, when Abeta-metal ion complexes undergo aggregation, they significantly lose their protective function and allow oxidative damage to occur.     

Pathways of dopamine oxidation mediated by nitric oxide

This study was aimed at establishing the interaction between dopamine and nitric oxide and elucidating the mechanistic aspects inherent in this interaction. At high (*) NO concentrations (microM range), dopamine underwent nitrosation with subsequent nitration. Nitrosation is proposed to occur via a nucleophilic attack to N(2)O(3) by dopamine. At low (*) NO concentrations (microM range), dopaminochrome was formed. EPR spin stabilization studies showed the occurrence of two o-semiquinone intermediates during dopaminochrome formation. Heats of formation obtained by AM1 semiempirical calculations supported the formation of the two o-semiquinone species. Hydroxyl radicals were detected by spin trapping EPR, and experiments performed with superoxide dismutase and catalase suggested that peroxynitrite was the source of HO(*). A mechanism is presented that considers the several factors influencing these reactions.     

Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells

Hydroxyl radicals (.OH) and superoxide anion radicals (O2.-) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44 degrees C, 30 min) treatment. Rate constants of the reactions between antioxidants and .OH or O2.- were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for .OH or O2.- was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for .OH or O2.-. The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia- induced apoptosis.     

Nordihydroguaiaretic acid is a potent in vitro scavenger of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion and hypochlorous acid and prevents in vivo ozone-induced tyrosine nitration in lungs

The antioxidant nordihydroguaiaretic acid (NDGA) has recently become well known as a putative anticancer drug. In this paper, it was evaluated the in vitro peroxynitrite (ONOO(-)), singlet oxygen ((1)O(2)), hydroxyl radical (OH(v)), hydrogen peroxide (H(2)O(2)), superoxide anion and hypochlorous acid (HOCl) scavenging capacity of NDGA. It was found that NDGA scavenges: (a) ONOO(-) (IC(50) = 4 +/- 0.94 microM) as efficiently as uric acid; (b) (1)O(2) (IC(50) = 151 +/- 20 microM) more efficiently than dimethyl thiourea, lipoic acid, N-acetyl-cysteine and glutathione; (c) OH(v) (IC(50) = 0.15 +/- 0.02 microM) more efficiently than dimethyl thiourea, uric acid, trolox, dimethyl sulfoxide and mannitol, (d) (IC(50) = 15 +/- 1 microM) more efficiently than N-acetyl-cysteine, glutathione, tempol and deferoxamine and (e) HOCl (IC(50) = 622 +/- 42 microM) as efficiently as lipoic acid and N-acetyl-cysteine. NDGA was unable to scavenge H(2)O(2). In an in vivo study in rats, NDGA was able to prevent ozone-induced tyrosine nitration in lungs. It is concluded that NDGA is a potent in vitro scavenger of ONOO(-), (1)O(2), OH(v), and HOCl and is able to prevent lung tyrosine nitration in vivo.     

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.     

Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 - ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 μM for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.     

Protective effect of histidine on para-nonylphenol-enhanced hydroxyl free radical generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum.

The present study examined the antioxidant effect of histidine, a singlet oxygen ((1)O(2)) scavenger, on para-nonylphenol (an environmental estrogen-like chemical)-enhanced hydroxyl radical (.OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in extracellular fluid of rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Introduction of para-nonylphenol (10 microM) significantly enhanced MPP+ -induced.OH generation. Histidine (25 mM) decreased the para-nonylphenol-enhanced.OH formation. Although the level of MPP+ -induced.OH formation trapped as DHBA after para-nonylphenol treatment increased, para-nonylphenol failed to increase either the level of dopamine and DHBA formation in the reserpinized animals. These results indicate that para-nonylphenol and MPP+ -enhanced.OH generation was based on 1O(2) production, and histidine may have a preventive effect on para-nonylphenol and MPP+ -induced.OH generation in rat striatum.     

E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide

The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low K(M) value of 10 microM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in k(cat)/K(MetSO). Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.