Antioxidant Activity of Caffeic Acid against Iron-Induced Free Radical Generation--A Chemical Approach.

Caffeic acid (CA) is a phenolic compound widely found in coffee beans with known beneficial effects in vivo. Many studies showed that CA has anti-inflammatory, anti-mutagenic, antibacterial and anti-carcinogenic properties, which could be linked to its antioxidant activity. Taking in consideration the reported in vitro antioxidant mechanism of other polyphenols, our working hypothesis was that the CA antioxidant activity could be related to its metal-chelating property. With that in mind, we sought to investigate the chemical antioxidant mechanism of CA against in vitro iron-induced oxidative damage under different assay conditions. CA was able to prevent hydroxyl radical formation promoted by the classical Fenton reaction, as determined by 2-deoxyribose (2-DR) oxidative degradation and DMPO hydroxylation. In addition to its ability to prevent hydroxyl radical formation, CA had a great inhibition of membrane lipid peroxidation. In the lipid peroxidation assays CA acted as both metal-chelator and as hydrogen donor, preventing the deleterious action promoted by lipid-derived peroxyl and alkoxyl radicals. Our results indicate that the observed antioxidant effects were mostly due to the formation of iron-CA complexes, which are able to prevent 2-DR oxidation and DMPO hydroxylation. Noteworthy, the formation of iron-CA complexes and prevention of oxidative damage was directly related to the pH of the medium, showing better antioxidant activity at higher pH values. Moreover, in the presence of lipid membranes the antioxidant potency of CA was much higher, indicating its enhanced effectiveness in a hydrophobic environment. Overall, our results show that CA acts as an antioxidant through an iron chelating mechanism, preventing the formation of free hydroxyl radicals and, therefore, inhibiting Fenton-induced oxidative damage. The chemical properties of CA described here--in association with its reported signaling effects--could be an explanation to its beneficial effects observed in vivo.

Superoxide dismutase 1-mediated production of ethanol- and DNA-derived radicals in yeasts challenged with hydrogen peroxide: molecular insights into the genome instability of peroxiredoxin-null strains.

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1Deltatsa2Delta) are more resistant to hydrogen peroxide than wild-type (WT) cells and consume it faster under fermentative conditions. Also, tsa1Deltatsa2Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1Deltatsa2Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu,Zn-superoxide dismutase (Sod1), whose expression and activity increased approximately 5- and 2-fold, respectively, in tsa1Deltatsa2Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1Deltatsa2Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1Deltatsa2Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

In vitro photodynamic activity of chloro(5,10,15,20-tetraphenylporphyrinato)indium(III) loaded-poly(lactide-co-glycolide) nanoparticles in LNCaP prostate tumour cells.

In(III)-meso-tetraphenylporphyrin (InTPP) was encapsulated into nanoparticles (smaller than 200 nm) of poly(d,l-lactide-co-glycolide) (PLGA) using the emulsification-evaporation technique. The photodynamic efficacy of InTPP-loaded nanoparticles and its cellular uptake was investigated with LNCaP prostate tumour cells, in comparison with the free InTPP. The effects of incubation time (1-3h), drug concentration (1.8-7.7 micromol/L) and incident light dose (15-45 J/cm(2)) with both encapsulated and free InTPP were studied. The type of cell death induced by the photochemical process using both encapsulated and free InTPP was also investigated. Cell viability was reduced more significantly with increasing values of these effects for InTPP-loaded nanoparticles than with the free drug. The cellular death induced by both encapsulated and free InTPP was preponderantly apoptotic. Confocal laser scanning microscopy data showed that the InTPP-loaded nanoparticles, as well free InTPP, were localized in the cells, and always in the perinuclear region. Encapsulated InTPP was measured by the intensity of fluorescence intensity of cell extracts and was three times more internalized into the cells than was the free InTPP. Electron paramagnetic resonance experiments corroborated the participation of singlet oxygen in the photocytotoxic effect of nanoparticles loaded with InTPP.

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis.

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.

Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics.

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.

Linoleic acid hydroperoxide reacts with hypochlorous acid, generating peroxyl radical intermediates and singlet molecular oxygen.

The reaction of hypochlorous acid (HOCl) with hydrogen peroxide is known to generate stoichiometric amounts of singlet molecular oxygen [O2 (1Deltag)]. This study shows that HOCl can also react with linoleic acid hydroperoxide (LAOOH), generating O2 (1Deltag) with a yield of 13 +/- 2% at physiological pH. Characteristic light emission at 1,270 nm, corresponding to O2 (1Deltag) monomolecular decay, was observed when HOCl was reacted with LAOOH or with liposomes containing phosphatidylcholine hydroperoxides, but not with cumene hydroperoxide or tert-butyl hydroperoxide. The generation of O2 (1Deltag) was confirmed by the acquisition of the spectrum of the light emitted in the near-infrared region showing a band with maximum intensity at 1,270 nm and by the observation of the enhancing effect of deuterium oxide and the quenching effect of sodium azide. Mechanistic studies using 18O-labeled linoleic acid hydroperoxide (LA18O18OH) showed that its reaction with HOCl yields 18O-labeled O2 (1Deltag) [18O2 (1Deltag)], demonstrating that the oxygen atoms in O2 (1Deltag) are derived from the hydroperoxide group. Direct analysis of radical intermediates in the reaction of LAOOH with HOCl by continuous-flow electron paramagnetic resonance spectroscopy showed a doublet signal with a g-value of 2.014 and a hyperfine coupling constant from the alpha-hydrogen of a(H) = 4.3 G, indicating the formation of peroxyl radicals. Taken together, our results clearly demonstrate that HOCl reacts with biologically relevant lipid hydroperoxides, generating O2 (1Deltag). In addition, the detection of 18O2 (1Deltag) and peroxyl radicals strongly supports the involvement of a Russell mechanism in the generation of O2 (1Deltag).

Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase.

The localization of Cu,Zn-superoxide dismutase in the mitochondrial intermembrane space suggests a functional relationship with superoxide anion (O2*-) released into this compartment. The present study was aimed at examining the functionality of Cu,Zn-superoxide dismutase and elucidating the molecular basis for its activation in the intermembrane space. Intact rat liver mitochondria neither scavenged nor dismutated externally generated O2*-, unless the mitochondrial outer membrane was disrupted selectively by digitonin. The activation of the intermembrane space Cu,Zn-superoxide dismutase following the disruption of mitochondrial outer membrane was largely inhibited by bacitracin, an inhibitor of protein disulphide-isomerase. Thiol alkylating agents, such as N-methylmaleimide or iodoacetamide, decreased intermembrane space Cu,Zn-superoxide dismutase activation during, but not after, disruption of the outer membrane. This inhibitory effect was overcome by exposing mitochondria to low micromolar concentrations of H2O2 before disruption of the outer membrane in the presence of the alkylating agents. Moreover, H2O2 treatment alone enabled intact mitochondria to scavenge externally generated O2*-. These findings suggest that intermembrane space Cu,Zn-superoxide dismutase is inactive in intact mitochondria and that an oxidative modification of its critical thiol groups is necessary for its activation.

Leucoisoprenochrome-o-semiquinone formation in freshly isolated adult rat cardiomyocytes.

Sustained high levels of circulating catecholamines can lead to cardiotoxicity. There is increasing evidence that this process may result from metal-catalyzed catecholamine oxidation into semiquinones, quinones, and aminochromes. We have previously shown that Cu2+-induced oxidation of isoproterenol into isoprenochrome induces toxic effects in isolated cardiomyocytes. The aim of this study was to characterize the isoproterenol oxidation process and to locate the formation of semiquinone radicals in cardiomyocyte suspensions. Freshly isolated rat cardiomyocytes were incubated with 1 or 10 mM isoproterenol and 20 microM Cu2+ for 4 h. The formation of an isoproterenol oxidation radical was detected in the extracellular medium, cells, membranes, and heavy organelles by electron spin resonance spectroscopy. An electron spin resonance signal assigned to leucoisoprenochrome-o-semiquinone increased in a time-dependent manner in the extracellular medium. A second electron spin resonance signal, characteristic of an immobilized radical, was also found in the cardiomyocytes. The latter was attributed to leucoisoprenochrome-o-semiquinone immobilized on cellular components such as membranes, cytoskeleton, nucleus, and heavy organelles. In addition, the levels of leucoisoprenochrome-o-semiquinone decreased in the presence of glutathione. Computer simulations of the experimental spectra indicate the formation of two distinct isomeric leucoisoprenochrome-o-semiquinone radicals during isoproterenol oxidation. The present study shows that the isoproterenol oxidation in isolated rat cardiomyocytes correlates with the formation of leucoisoprenochrome-o-semiquinone in the cells and in the extracellular medium, suggesting that it might be involved in cardiotoxicity induced by the oxidation of catecholamines.

Usnic acid-induced necrosis of cultured mouse hepatocytes: inhibition of mitochondrial function and oxidative stress.

Usnic acid, a lichen acid, is a compound found in crude medicines and dietary supplements, including Lipokinetix, a supplement marketed as a weight loss agent that caused hepatotoxicity and acute liver failure in patients. In this study, we examined the toxicity of usnic acid and assessed whether usnic acid may be contributing to hepatotoxicity caused by Lipokinetix. In primary cultured murine hepatocytes, usnic acid treatment (5 microM) resulted in 98% necrosis within 16 hr (no apoptosis was detected). Usnic acid treatment was associated with early inhibition and uncoupling of the electron transport chain in mitochondria of cultured hepatocytes. This inhibition of mitochondria by usnic acid corresponded with a fall in ATP levels in hepatocytes. In isolated liver mitochondria, usnic acid was observed to directly inhibit and uncouple oxidative phosphorylation. Oxidative stress appears to be central in usnic acid-induced hepatotoxicity based on the following findings: (1) pretreatment with antioxidants (butylated hydroxytoluene+Vitamin E) decreased usnic acid-induced necrosis by nearly 70%; (2) depletion of mitochondrial GSH with diethylmaleate increased susceptibility of hepatocytes to usnic acid; (3) usnic acid treatment was associated with increase free radical generation, measured using the fluorescent probe, dichlorodihydrofluorescin. The source of reactive oxygen species after usnic acid treatment include autoxidation of usnic acid and increased hydrogen peroxide generation by mitochondria caused by usnic acid inhibition of the respiratory chain, with the latter playing a more prominent role. Taken together, our results suggest that usnic acid is a strong hepatotoxic agent that triggers oxidative stress and disrupts the normal metabolic processes of cells. Usnic acid therefore may contribute to the hepatotoxic effects of Lipokinetix and its use in any supplement must come into question.

Effect of glutathione depletion on sites and topology of superoxide and hydrogen peroxide production in mitochondria.

In this work, the topology of mitochondrial O2(-)(radical) and H2O2 generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases O2(-)(radical) into the matrix (where it is converted to H2O2 by Mn-SOD) but not into the intermembrane space. No free radical generation was observed from complex II, but succinate treatment caused H2O2 generation from the matrix through a reverse electron flow to complex I. Complex III was found to release O2(-)(radical) into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of UQO>- (ubisemiquinone at the Qo site) in complex III, enhanced both H2O2 generation from the matrix and O2(-)(radical) production from the intermembrane space. On the other hand, myxothiazol, which inhibits UQO>- formation, completely inhibited antimycin induced O2(-)(radical) toward the intermembrane space and inhibited H2O2 generation from the matrix by 70%. However, myxothiazol alone enhanced H2O2 production from complex III, suggesting that other components of complex III besides the UQO- can cause O2(-)(radical) generation toward the matrix. As expected, mitochondrial GSH was found to modulate H2O2 production from the matrix but not O2- generation from the intermembrane space. Low levels of GSH depletion (from 0-40%, depending on the rate of H2O2 production) had no effect on H2O2 diffusion from mitochondria. Once this GSH depletion threshold was reached, GSH loss corresponded to a linear increase in H2O2 production by mitochondria. The impact of 50% mitochondrial GSH depletion, as seen in certain pathological conditions in vivo, on H2O2 production by mitochondria depends on the metabolic state of mitochondria, which governs its rate of H2O2 production. The greater the rate of H2O2 generation the greater the effect 50% GSH depletion had on enhancing H2O2 production.

Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol.

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O(2)*-) and hydrogen peroxide (H(2)O(2)). The electron transfer chain of mitochondria is a well documented source of H(2)O(2); however, the release of O(2)*- from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O(2)*-, elucidating the mechanisms underlying the release of O(2)*- from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O(2)*-. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O(2)*- release from mitochondria were estimated at approximately 0.04 nmol/min/mg protein, a value increased approximately 8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O(2)*- production from mitochondria by approximately 55%, thus suggesting that a large portion of O(2)*- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O(2)*- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells.

Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (*NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. *NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on *NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of *NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between *NO and dopamine was not observed at *NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from *NO damage. Dopamine oxidation by *NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by *NO is higher. These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of *NO. In the early stages of Parkinson's disease, *NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of *NO, which involve irreversible impairment of mitochondrial respiration.

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.