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Summary: Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However, it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and reactive oxygen species (ROS) generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial-targeted antioxidants inhibited adipocyte differentiation, which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed that ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation but are a causal factor in promoting adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Protein tyrosine oxidation mechanisms in hydrophobic biocompartments (i.e., biomembranes, lipoproteins) leading to nitrated, dimerized, and hydroxylated products are just starting to be appreciated. This chapter reports on the use of the hydrophobic tyrosine analog N‐t‐BOC‐l‐tyrosine tert‐butyl ester (BTBE) incorporated to phosphatidyl choline liposomes to study peroxynitrite‐dependent tyrosine oxidation processes in model biomembranes. The probe proved to be valuable in defining the role of biologically relevant variables in the oxidation process, including the action of hydrophilic and hydrophobic peroxynitrite and peroxynitrite‐derived free radical scavengers, transition metal catalysts, carbon dioxide, molecular oxygen, pH, and fatty acid unsaturation degree. Moreover, detection of the BTBE phenoxyl radical and relative product distribution yields of 3‐nitro‐, 3,3′‐di‐, and 3‐hydroxy‐BTBE in the membrane fully accommodate with a free radical mechanism of tyrosine oxidation, with physical chemical and biochemical determinants that in several respects differ of those participating in aqueous environments. The methods presented herein can be extended to explore the reaction mechanisms of tyrosine oxidation by other biologically relevant oxidants and in other hydrophobic biocompartments. [Copyright &y& Elsevier]

Abstract: Using both high‐performance liquid chromatography (HPLC) and electron spin resonance (ESR) spin‐trappng techniques, we developed an analytical methodology for investigating intramolecular electron transfer‐mediated tyrosyl nitration and cysteine nitrosation in model peptides. Peptides N‐acetyl‐TyrCys‐amide (YC), N‐acetyl‐TyrAlaCys‐amide, N‐acetyl‐TyrAlaAlaCys‐amide, and N‐acetyl‐TyrAlaAlaAlaAlaCys‐amide were used as models. Product analysis showed that nitration and oxidation products derived from YC and related peptides in the presence of myeloperoxidase (MPO)/H2O2/NO2 − were not detectable. The major product was determined to be the corresponding disulfide (e.g., YCysCysY), suggestive of a rapid electron transfer from the tyrosyl radical to the cysteinyl residue. ESR spin‐trapping experiments with 5,5′‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that thiyl radical intermediates were formed from peptides (e.g., YC) treated with MPO/H2O2 and MPO/H2O2/NO2 −. Blocking the thiol group in YC totally abrogated thiyl radical formation. Under similar conditions, we were, however, able to trap the tyrosyl radical using the spin trap dibromonitrosobenzene sulfonic acid (DBNBS). Competition spin‐trapping experiments revealed that intramolecular electron transfer is the dominant mechanism for thiyl radical formation in YC peptides. We conclude that a rapid intramolecular electron transfer mechanism between redox‐sensitive amino acids could influence both protein nitration and nitrosation reactions. This mechanism brings together nitrative, nitrosative, and oxidative mechanisms in free radical biology. [Copyright &y& Elsevier]

We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP+ and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP+) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP+ -induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed. [ABSTRACT FROM AUTHOR]

Previously, we showed that oxidation of tryptophan-32 (Trp-32) residue was crucial for H2O2/bicarbonate (HCO3𑁦-)dependent covalent aggregation of human Cu,Zn SOD1 (hSOD1). The carbonate anion radical (CO3𑁦-)induced oxidation of Trp-32 to kynurenine-type oxidation products was proposed to cause the aggregation of hSOD1. Here we used the matrix-assisted laser desorption ionization–time of flight mass spectroscopy, high-performance liquid chromatography–electrospray ionization mass spectroscopy, and liquid chromatography mass spectroscopy methods to characterize products. Results show that a peptide region (31–36) of hSOD1 containing the Trp-32 residue (VWGSIK) is oxidatively modified to the N-formylkynurenine (NFK)- and kynurenine (Kyn)-containing peptides (V(NFK)GSIK) and (V(Kyn)GSIK) during HCO−dependent peroxidase activity of hSOD1. Also, UV photolysis of a cobalt complex that generates authentic CO3𑁦- radical induced a similar product profile from hSOD1. Similar products were obtained using a synthetic peptide with the same amino acid sequence (i.e., VWGSIK). We propose a mechanism involving a tryptophanyl radical for CO3𑁦-induced oxidation of Trp-32 residue (VWGSIK) in hSOD1 to V(NFK)GSIK and V(Kyn)GSIK. [Copyright &y& Elsevier]

An abstract of the article "Hydrophobic Tyrosyl Probes for Monitoring Nitration Reactions in Membranes," by Hao Zhang and colleagues is presented.

There is much interest in the nitration and oxidation reaction mechanisms initiated by superoxide radical anion (O2⨪) and nitric oxide (̇NO). It is well known that O2⨪ and N0 rapidly react to form a potent oxidant, peroxynitrite anion (ONOO-). However, indirect measurements with the existing probes (e.g. dihydrorhodamine) previously revealed a bell-shaped response to co-generated ̇NO and O2⨪ fluxes, with the maximal yield of the oxidation or nitration product occurring at a 1:1 ratio. These results raised doubts on the formation of ONOO- per se at various fluxes of ̇NO and O2⨪. Using a novel fluorogenic probe, coumarin-7-boronic acid, that reacts stoichiometrically and rapidly with ONOO- (k = 1.1 × 106 M-1S-1), we report that ONOO- formation increased linearly and began to plateau after reaching a 1:1 ratio of co-generated ̇NO and O2⨪ fluxes. We conclude that ONOO- is formed as the primary intermediate during the reaction between ̇NO and O2⨪ co-generated at different fluxes. [ABSTRACT FROM AUTHOR]

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H2O2, and nitrite anion (NO2-) and with horseradish peroxidase and H2O2. Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Aala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAA C). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H2O2 and MPO/H2O2/NO2-. The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH3) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H2O2/NO2-. However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H2O2/NO2-/YCSSCH3 system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H2O2/̇NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed. [ABSTRACT FROM AUTHOR]

In this review, we describe the free radical mechanism of covalent aggregation of human copper, zinc superoxide dismutase (hSOD1). Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 mediated by the SOD1 peroxidase-dependent formation of carbonate radical anion (CO3⋅-), a potent and selective oxidant. This species presumably diffuses out the active site of hSOD1 and reacts with tryptophan residue located on the surface of hSOD1. The oxidative degradation of tryptophan to kynurenine and N-formyl kynurenine results in the covalent crosslinking and aggregation of hSOD1. Implications of oxidant-mediated aggregation of hSOD1 in the increased cytotoxicity of motor neurons in amyotrophic lateral sclerosis are discussed. [Copyright &y& Elsevier]

Studies the bicarbonate-dependent peroxidase activity of human copper, zinc-superoxide dismutase (SOD). Hydroxylation and oxidation of nitrone spin traps by bicarbonate-dependent peroxidase activity; Effect of amino acids on the formation of human SOD aggregation; Effect of spin traps on the aggregation of human SOD.

Recently, we demonstrated that dimeric apocynin prevented loss of motor function in the leucine-rich repeat kinase 2 (LRRK2 R1441G ) transgenic (tg) mouse (treated with 200 mg/kg, three times per week) [B.P. Dranka et al., Neurosci. Lett. 549 (2013) 57–62]. Here we extend those studies by treating LRRK2 R1441G mice with an orally-available, mitochondrially-targeted apocynin derivative. We hypothesized that the increased mitochondrial permeability of Mito-apocynin, due to the triphenylphosphonium moiety, would allow improvement of Parkinson's disease (PD) symptoms at lower doses than those required for diapocynin. Tests of motor coordination (pole test, Rotor-Rod) revealed a significant deficit in coordinated motor function in LRRK2 R1441G mice by 15 months of age. Decreased performance on the pole test and Rotor-Rod in the LRRK2 R1441G mice was prevented with Mito-apocynin treatment (3 mg/kg, three times per week). Decreased olfactory function is an early indication of PD in human patients. LRRK2 R1441G tg mice displayed deficits in sense of smell in both the hidden treat test, and a radial arm maze test. Interestingly, treatment with Mito-apocynin prevented this hyposmia, and animals retained normal ability to identify either a scented treat or a food pellet as well as wild type littermates. Together, these data demonstrate that the mitochondria-targeted apocynin analog is effective in preventing early PD-like symptoms in the LRRK2 R1441G mouse model. [ABSTRACT FROM AUTHOR]

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7-23 M-1 s-1) were significantly higher than that measured for H2O2 (1.5 M-1 s-1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1-1.5×103 M-1 s-1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. [ABSTRACT FROM AUTHOR]

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate readerbased fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate readerbased oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action. [ABSTRACT FROM AUTHOR]

Highlights: [•] LRRK2R1441G mice lose coordinated movement without gross motor deficits. [•] Diapocynin prevents deficits in motor coordination in LRRK2R1441G mice. [•] LRRK2R1441G mice may be useful for modeling non-motor PD symptoms. [Copyright &y& Elsevier]

Abstract: Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr+), the reduction product of propidium iodide, for detecting extracellular superoxide (O2 •−). HPr+ is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O2 •−. We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr+ and allow for exclusive detection of extracellular O2 •−. Our results indicate that O2 •− reacts with HPr+ (k=1.2×104 M−1 s−1) to form exclusively 2-hydroxypropidium (2-OH-Pr2+) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O2 •− (Zhao et al., Free Radic. Biol. Med. 34:1359–1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O2 •− with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O2 •− and HPr+ is slightly higher than that of HE and O2 •− and is closer to that of Mito-HE and O2 •−. Similar to HE, HPr+ undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fenton''s reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr2+) and the dimeric products (e.g., Pr2+-Pr2+). In contrast to HE, there was very little intracellular uptake of HPr+. We conclude that HPr+ is a useful probe for detecting O2 •− and other one-electron oxidizing species in an extracellular milieu. [Copyright &y& Elsevier]

Abstract: Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia–reperfusion (I/R) injury; however, its exact role and its spatial–temporal relationship with inflammation are elusive. Herein we explore the spatial–temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia–reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage. [Copyright &y& Elsevier]

Alcohol treatment induces oxidative stress by a combination of increased production of partially reduced oxygen species and decreased cellular antioxidant pool, including GSH. Recently, we showed that mitochondrion-targeted CYP2E1 augments alcohol-mediated toxicity, causing an increase in reactive oxygen species production and oxidative stress. Here, we show that cytochrome c oxidase (CcO), the terminal oxidase of the mitochondrial respiratory chain, is a critical target of CYP2E1-mediated alcohol toxicity. COS-7 and Hep G2 cell lines expressing predominantly mitochondrion-targeted (Mt++) CYP2E1 and livers from alcohol-treated rats showed loss of CcO activity and increased protein carbonylation, which was accompanied by a decline in the steady state levels of subunits I, IVI1, and Vb of the CcO complex. This was also accompanied by reduced mitochondrial DNA content and reduced mitochondrial mRNA. These changes were more prominent in Mt++ cells in comparison with wild type (WT) CYP2E1-expressing or ER+ (mostly microsome-targeted) cells. In addition, mitochondrion-specific antioxidants, ubiquinol conjugated to triphenyl phosphonium, triphenylphosphonium conjugated carboxyl proxyl, and the CYP2E1 inhibitor diallyl sulfide prevented the loss of CcO activity and the CcO subunits, most likely through reduced oxidative damage to the enzyme complex. Our results suggest that damage to CcO and dissociation of respirosome complexes are critical factors in alcohol-induced toxicity, which is augmented by mitochondrion-targeted CYP2E1. We propose that CcO is one of the direct and immediate targets of alcohol-induced toxicity causing respiratory dysfunction. [ABSTRACT FROM AUTHOR]

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of antiinflammatory antioxidants. [ABSTRACT FROM AUTHOR]

Abstract: Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatin''s antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity. [Copyright &y& Elsevier]

Abstract: The objective of this study was to assess the neuroprotective effects of a mitochondria-targeted antioxidant, Mito-Q10, the coenzyme-Q analog attached to a triphenylphosphonium cation that targets the antioxidant to mitochondria, in experimental models of Parkinson''s disease (PD). Primary mesencephalic neuronal cells and cultured dopaminergic cells were treated with 1-methyl-4-phenylpyridinium (MPP+), an active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and mice were used for testing the efficacy of Mito-Q10. MPP+ treatment caused a dose-dependent loss of tyrosine hydroxylase and membrane potential and an increase in caspase-3 activation in dopaminergic cells, which were reversed by Mito-Q10. MPTP treatment induced a loss of striatal dopamine and its metabolites, inactivation of mitochondrial aconitase in the substantia nigra, and a loss of locomotor activity in mice. Treatment with Mito-Q10 significantly inhibited both MPP+- and MPTP-induced neurotoxicity in cell culture and mouse models. Collectively, these results indicate that mitochondrial targeting of antioxidants is a promising neuroprotective strategy in this preclinical mouse model of PD. [Copyright &y& Elsevier]

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q10. Inhibition of cancer cell growth by MitoQ was associated with G1/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G1 cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO−) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO−, hypochlorous acid (HOCl), and hydrogen peroxide (H2O2) and found that ONOO− reacts with 4-acetylphenylboronic acid nearly a million times (k =1.6×106 M−1 s−1) faster than does H2O2 (k =2.2 M−1 s−1) and over 200 times faster than does HOCl (k =6.2×103 M−1 s−1). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO− formed in cellular and cell-free systems. [Copyright &y& Elsevier]

Abstract: It has been proposed that autoxidation of nitric oxide ( NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of NO/O2. The transmembrane cysteinyl peptides C4 (AcNH-KKACALA(LA)6KK-CONH2) and C8 (AcNH-KKALALACALA(LA)3KK-CONH2) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C8 and C4, and glutathione (GSH; 300 μM) were treated with a NO donor, DEA-NONOate, and nitrosothiol formation was determined under various O2 levels. Surprisingly, the more hydrophobic cysteinyl peptide, C8, did not yield any S-nitrosated product compared to GSH in the aqueous phase or C4 peptide in the liposomes in the presence of NO/O2. These data suggest that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of NO/O2. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H2O2 and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H2O2- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H2O2- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of 55Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the “untargeted” carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function. [Copyright &y& Elsevier]

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H2O2 and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 μM) and MitoVit-E (1 μM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H2O2- and lipid peroxide-induced 2′,7′-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H2O2- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of 55Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis. [ABSTRACT FROM AUTHOR]

Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-α, hy- poxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide ('NO) during hydrogen peroxide (H2O2)-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C2-ceramide (C2-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C2-cer (5–20 μM) to BAECs enhanced 'NO generation. However, at higher concentrations of C2-cer (≥20 μM), NO generation did not increase proportionately. C2-cer (20–50 μM) also resulted in H2O2-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of 55Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. Nw-Nitro-L-arginine methyl ester (L-NAME), a nonspecific inhibitor of nitric-oxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C2-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C2-cer (≤10 μM) but was greatly suppressed at higher concentrations (>10 μM). Intracellular scavengers of H2O2, cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C2-cer- and/or L-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C2-cer-induced H2O2 and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that 'NO exerts an antioxidative and cytoprotective role. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX), a widely used chemotherapeutic agent, exhibits cardiotoxicity as an adverse side effect in cancer patients. DOX-mediated cardiomyopathy is linked to its ability to induce apoptosis in endothelial cells and cardiomyocytes by activation of p53 protein and reactive oxygen species. We evaluated the potential roles of H2O2 and p53 in DOX-induced apoptosis in normal bovine aortic endothelial cells and adult rat cardiomyo. cytes and in tumor cell lines PA-1 (human ovarian teratocarcinoma) and MCF-7 (human breast adenocareinoma). Time course measurements indicated that activation of caspase-3 preceded the stimulation of p53 transcriptional activity in endothelial cells. In contrast, DOX caused early activation of p53 in tumor cells that was followed by caspase-3 activation and DNA fragmenta. tion. These findings suggest that the transcriptional activation of p53 in DOX-induced apoptosis in endothelial cells may not be as crucial as it is in tumor cells. Further evidence was obtained using a p53 inhibitor, pifithrin-α. Pifithrin-α completely suppressed DOX-induced activation of p53 in both normal and tumor cell lines and prevented apoptosis in tumor cell lines but not in endothelial cells and cardiomyocytes. In contrast, detoxification of H2O2, either by redox-active metalloporphyrin or overexpression of glutathione peroxidase, decreased DOX-induced apoptosis in endothelial cells and cardiomyocytes but not in tumor cells. This newly discovered mechanistic difference in DOX-induced apoptotic cell death in normal versus tumor cells will be useful in developing drugs that selectively mitigate the toxic side effects of DOX without affecting its antitumor action. [ABSTRACT FROM AUTHOR]

1-Methyl-4-phenylpyridinium (MPP+) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP+-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of α-synuclein (α-syn) in MPP+-induced oxidative stress and apoptosis. Results show that MPP+ inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H2O2 generation, TfR expression and α-syn expression/aggregation. Pretreatment with cellpermeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, α-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing α-syn exacerbated MPP+ toxicity, whereas antisense α-syn treatment totally abrogated MPP+-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of α-syn in MPP+-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP+induced iron signaling is responsible for intracellular oxidant generation, α-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed. [ABSTRACT FROM AUTHOR]

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO3 > OH > CH3, while in cell suspensions it was SO3 > OH ≈ CH3. The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study. [Copyright &y& Elsevier]

Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500–530 nm; emission, 590–620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed. [Copyright &y& Elsevier]

Studies the transmembrane nitration of hydrophobic tyrosyl peptides. Comparison between the amount of the hydrophobic tyrosyl probe and the tyrosine in the aqueous phase; Dependency of tyrosine nitration to the intermembrane location of the tyrosine probes; Initiation of nitration by slow infusion of peroxynitrite.

The cytoprotective effects of redox-active metalloporphyrins (e.g., FeTBAP and MnTBAP) were generally attributed to their ability to scavenge reactive oxygen and nitrogen species. In this study, we evaluated the pro- and antiapoptotic potentials of different metalloporphyrins containing iron, cobalt, zinc, and manganese in adult rat cardiomyocytes exposed to doxorubicin (DOX), an anticancer drug that forms superoxide and hydrogen peroxide via redox-cycling of DOX semiquinone in the presence of molecular oxygen. We used electron spin resonance/spin trapping and cytochrome c reduction to assess the scavenging of superoxide anion by metalloporphyrins. Superoxide anion was effectively scavenged by FeTBAP and MnTBAP but not by CoTBAP and ZnTBAP. FeTBAP efficiently scavenged H2O2. Both CoTBAP and FeTBAP inhibited DOX-induced cardiomyocyte apoptosis. These findings implicate that mechanisms other than oxy-radical scavenging may account for their antiapoptotic property. In addition, CoTBAP and FeTBAP induced heme oxygenase-1 more potently than did MnTBAP and ZnTBAP. Inhibition of heme oxygenase abolished the protective effect of CoTBAP and reduced the protection by FeTBAP against DOX-induced cardiomyocyte apoptosis. We propose that metalloporphyrins can inhibit apoptosis either by inducing heme oxygenase-1 and antiapoptotic protein signaling or by scavenging reactive oxygen species. [Copyright &y& Elsevier]