On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase.

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.     

The role of hydroxyl radicals in tetrachlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts

Tetrachlorohydroquinone (TCHQ), which has previously been identified as a metabolite of pentachlorophenol, induces DNA strand breaks in isolated DNA and in human fibroblasts. Strand break formation in PM2 DNA is prevented by the addition of catalase and the hydroxyl radical scavengers DMSO, ethanol and mannitol, whereas addition of SOD reduced SSB only slightly. Oxygen radicals are formed by the autoxidation of TCHQ to the tetrachlorosemiquinone radical. Desferrioxamine (0.2 mM) completely abolished strand break formation, whereas the metal chelator DETAPAC (1 mM) reduced SSB by only 8.5%. The formation of the semiquinone radical at physiological conditions is shown by ESR spectroscopy. Exposure of human fibroblasts to TCHQ also leads to DNA single strand breaks measured by the alkaline elution assay. These were reduced by addition of 5% DMSO. This indicates that at least part of the strand break formation in human cells is also due to the action of hydroxyl radicals.     

Thiol oxidation induced by oxidative action of adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.     

Oxidation of 4-methylcatechol: implications for the oxidation of catecholamines

At alkaline pH, 4-methylcatechol oxidizes more rapidly than the related catecholamines: dopamine, norepinephrine, and epinephrine. This oxidation is not inhibited by superoxide dismutase or catalase, indicating that O2 itself is the oxidant, but the reduction potential of O2/O2-* is too low for it to oxidize 4-methylcatechol directly. Instead, O2 oxidizes the 4-methylcatechol semiquinone, which is formed by comproportionation of 4-methylcatechol and its o-quinone. Aniline reacts very quickly with the o-quinone and thus stops the comproportionation reaction that oxidizes 4-methylcatechol to the semiquinone. Oxidation of 4-methylcatechol then requires superoxide, and in the presence of aniline, oxidation of 4-methylcatechol by O2 is inhibited by superoxide dismutase. When catecholamines oxidize, the side chain amine inserts into the catechol o-quinone, forming a bicyclic compound. By eliminating the quinone, this ring closure prevents comproportionation and the consequent oxidation of catecholamines by O2. It also prevents reaction of the quinone with other compounds and the formation of potentially toxic products.     

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).     

The DNA cleavage induced by a chromium(V) complex and by chromate and glutathione is mediated by activated oxygen species

The number of strand breaks induced by the combination of chromate and glutathione (GSH) in PM2 DNA was effectively reduced upon addition of the hydroxyl radical scavengers dimethyl sulphoxide (DMSO), formate and benzoate. Administration of catalase also led to a depression of DNA degradation whereas superoxide dismutase (SOD) had very little influence. Essentially the same results were obtained in experiments employing a chromium(V) complex Na4(GSH)4Cr.8H20, which is an intermediate chromium species isolated from the reduction of chromate by glutathione. DNA cleavage was dependent on the presence of iron (FeCl3). When compared with the number of breaks produced by FeCl3 and GSH alone, chromate stimulated the generation of single-strand breaks. These findings suggest that hydroxyl radicals are one ultimate DNA cleaving agent in both reactions. A reaction scheme for the production of hydroxyl radicals is proposed.     

Thiol Oxidation Induced by Oxidative Action of Adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Approaches to Prevent Dopamine Quinone-Induced Neurotoxicity

Dopamine (DA) and its metabolites containing two hydroxyl residues exert cytotoxicity in dopaminergic neuronal cells, primarily due to the generation of highly reactive DA and DOPA quinones. Quinone formation is closely linked to other representative hypotheses such as mitochondrial dysfunction, inflammation, oxidative stress, and dysfunction of the ubiquitin-proteasome system, in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease and methamphetamine-induced neurotoxicity. Therefore, pathogenic effects of the DA quinone have focused on dopaminergic neuron-specific oxidative stress. Recently, various studies have demonstrated that some intrinsic molecules and several drugs exert protective effects against DA quinone-induced damage of dopaminergic neurons. In this article, we review recent studies on some neuroprotective approaches against DA quinone-induced dysfunction and/or degeneration of dopaminergic neurons. Special issue article in honor of Dr. Akitane Mori.     

Quinone toxicity in hepatocytes without oxidative stress

The toxicity of quinones is believed to be mediated via redox cycling involving formation of semiquinone radicals which autoxidize to form active oxygen species. However, when the cytotoxicity of benzoquinones was compared using freshly isolated rat hepatocytes, benzoquinones which did not mediate oxidative stress were highly toxic. Thus, the benzoquinone analogs in decreasing order of cytotoxicity were 2-CH3-, 2-Br-, unsubstituted, 2,6-(CH3)2-, 2,5-(CH3)2-, and 2,3,5-(CH3)3-benzoquinone. Cellular thiols were rapidly depleted and glutathione (GSH) was converted to a quinone conjugate without oxidation to glutathione disulfide. No increase in cyanide-resistant respiration was observed and benzoquinone-induced cytotoxicity was not enhanced by inactivation of catalase or glutathione reductase. In contrast, duroquinone [2,3,5,6-(CH3)4-benzoquinone], which stimulated cyanide-resistant respiration and GSH oxidation, was only cytotoxic when catalase or glutathione reductase was inactivated. These results suggest that alkylation and/or oxidative stress may be important mechanisms in the cytotoxicity of benzoquinone derivatives.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Cu(I)-glutathione complex: a potential source of superoxide radicals generation

Cu(2+) ions and GSH molecules interact swiftly to form the complex Cu(I)-glutathione. We investigated the potential capacity of such complex to reduce molecular oxygen. The addition of SOD to a solution containing Cu(I)-glutathione led to a sustained decline of the basal oxygen level. Such effect was partially reverted by the addition of catalase. The complex was able to induce the reduction of cytochrome c and the oxidation of dyhydroethidium into 2-hydroxyethidium. Both effects were totally blocked by SOD. The ability of the complex to generate superoxide radicals was confirmed by EPR spin-trapping. Cu(I)-glutathione induces no oxidation of fluorescein, a hydroxyl radical-sensitive probe. We conclude that in solutions containing the complex, oxygen is continually reduced into superoxide, and that-in absence of interceptors-the latter radicals are quantitatively re-oxidized into molecular oxygen. We suggest that, by functioning as a continuous source of superoxide, the complex could potentially affect a broad range of susceptible biological targets.     

Exploring by pulsed EPR the electronic structure of ubisemiquinone bound at the QH site of cytochrome bo3 from Escherichia coli with in vivo 13C-labeled methyl and methoxy substituents

The cytochrome bo(3) ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O(2) to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo(3) as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 (13)C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group (13)C-labeled. The (13)C-labeled quinone isolated from cytochrome bo(3) was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.     

Toxic and Protective Effects of l-DOPA on Mesencephalic Cell Cultures

Abstract: The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 μ M ) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [³H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 μ M ), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [³H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, l-DOPA prevented cell death by L-BSO.     

Characterisation of the PQQ cofactor radical in quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa by electron paramagnetic resonance spectroscopy

The binding pocket of the pyrroloquinoline quinone (PQQ) cofactor in quinoprotein alcohol dehydrogenases contains a characteristic disulphide ring formed by two adjacent cysteine residues. To analyse the function of this unusual structural motif we have investigated the wild-type and a double cysteine:alanine mutant of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa by electron paramagnetic resonance (EPR) spectroscopy. Thus, we have obtained the principal values for the full rhombic g-tensor of the PQQ semiquinone radical by high-field (94 GHz) EPR necessary for a discrimination of radical species in dehydrogenases containing PQQ together with other redox-active cofactors. Our results show that the characteristic disulphide ring is no prerequisite for the formation of the functionally important semiquinone form of PQQ.     

Superoxide dismutase enhanced the formation of hydroxyl radicals in a reaction mixture containing xanthone under UVA irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 microM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.).     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.     

Is There a Correlation Between In Vitro Antioxidant Potential and In Vivo Effect of Carvacryl Acetate Against Oxidative Stress in Mice Hippocampus?

This study investigated in vitro and in vivo antioxidant potential of carvacryl acetate (CA), a derivative of carvacrol, monoterpenic component of oregano. The correlation between in vitro and in vivo CA effects was also determined. In vitro tests measured thiobarbituric acid reactive species content, nitrite formation and hydroxyl radical levels. In vivo tests measured thiobarbituric acid reactive species content, nitrite concentration and reduced glutathione (GSH) levels, as well as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase activities were measured, using mice hippocampus. The CA administrations for in vivo tests were intraperitoneally and acutely improved. CA reduced lipid peroxidation, nitrite and hydroxyl radical contents in vitro as well as lipid peroxidation and nitrite content in vivo. It also increased reduced GSH levels and GPx as well as catalase activities. Moreover, CA required a lower concentration to inhibit 50 % of free radicals measured in vitro than trolox. There was significant negative correlation between in vitro nitrite levels and in vivo reduced GSH levels; in vitro nitrite content and in vivo GPx activity as well as in vitro hydroxyl radical levels and in vivo SOD activity. To date, this is the first study which suggests vitro and in vivo antioxidant potential to this monoterpene and the correlation between these parameters.