8-oxoguanine DNA glycosylase (Oggl) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Oggl is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the β-Oggl isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Oggl functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human α-hOggl (mt-hOggl) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial α-hOggl mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOggl in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase and apoptosis suggesting that the protective effects of mt-hOgg 1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOggl co-precipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOggl silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOggl acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.
DNA repair; aconitase; Oggl; free radicals; asbestos; mitochondria
Neurologic conditions including stroke, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are leading causes of death and long-term disability in the United States, and efforts to develop novel therapeutics for these conditions have historically had poor success in translating from bench to bedside. Hypoxia Inducible Factor-1alpha (HIF-1α) mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway are neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adpatation in detail, and provide perspective on which targets within this pathway appear to be ripest for preventing and repairing neurodegeneration. Further, we highlight the role of HIF prolyl hydroxylases as emerging targets for the salutary effects of metal chelators on ferroptosis in vitro as well in animal models of neurological diseases.
Metal chelators; neurodegeneration; hypoxia inducible factors; transcription; prolyl hydroxylases
Mitochondrial production of reactive oxygen species is often considered an unavoidable consequence of aerobic metabolism and currently cannot be manipulated without perturbing oxidative phosphorylation. Antioxidants are widely used to suppress effects of reactive oxygen species after formation, but they can never fully prevent immediate effects at the sites of production. To identify site-selective inhibitors of mitochondrial superoxide/H2O2 production that do not interfere with mitochondrial energy metabolism, we developed a robust small-molecule screen and secondary profiling strategy. We describe the discovery and characterization of a compound (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; CN-POBS) that selectively inhibits superoxide/H2O2 production from the ubiquinone-binding site of complex I (site IQ) with no effects on superoxide/H2O2 production from other sites or on oxidative phosphorylation. Structure/activity studies identified a core structure that is important for potency and selectivity for site IQ. By employing CN-POBS in mitochondria respiring on NADH-generating substrates, we show that site IQ does not produce significant amounts of superoxide/H2O2 during forward electron transport on glutamate plus malate. Our screening platform promises to facilitate further discovery of direct modulators of mitochondrially-derived oxidative damage and advance our ability to understand and manipulate mitochondrial reactive oxygen species production in both normal and pathological conditions.
superoxide; hydrogen peroxide; antioxidant; glycerol 3-phosphate dehydrogenase; NADH:Q oxidoreductase; complex II; complex III; energy metabolism; respiratory complexes
Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegen-erative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H2O2 or by the Fe(II)/ascorbate/H2O2 system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H2O2 relative to the Fe(II)/ascorbate/H2O2 system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H2O2 concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H2O2, it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA–cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.
RNA oxidation; Cytochrome c release; Cross-link; Abasic site; 8-Hydroxyguanosine; Free radicals
Nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase; NOX) is a complex enzyme responsible for increased levels of reactive oxygen species (ROS), superoxide (O2.−). NOX derived O2.− is a key player in oxidative stress and inflammation mediated multiple secondary injury cascades (SIC) following traumatic brain injury (TBI). The O2.− reacts with nitric oxide (NO), produces various reactive nitrogen species (RNS), and contributes to apoptotic cell death. Following a unilateral cortical contusion, young adult rats were killed at various times post injury (1, 3, 6, 12, 24, 48, 72, and 96 h). Fresh tissue from the hippocampus was analyzed for NOX activity, and level of O2.−. In addition we evaluated the translocation of cytosolic NOX proteins (p67Phox, p47Phox and p40Phox) to the membrane, along with total NO and the activation (phosphorylation) of endothelial nitric oxide synthase (p-eNOS). Results show that both enzymes and levels of O2.− and NO have time dependent injury effects in the hippocampus. Translocation of cytosolic NOX proteins into membrane, NOX activity and O2.− were also increased in a time dependent fashion. Both, NOX activity and O2.− were increased at 6 h. Levels of p-eNOS increased within 1 h, with significant elevation of NO at 12 h post TBI. Levels of NO failed to show a significant association with p-eNOS, but did associate with O2.−. NOX up-regulation strongly associated with both the levels of O2.− and also total NO. The initial 12 hours post TBI are very important as a possible window of opportunity to interrupt SIC. It may be important to selectively target the translocation of cytosolic subunits for the modulation of NOX function.
NADPH-oxidase; free radicals; secondary injury cascades; traumatic brain injury
Recently, Hall et al. reported that ethidium (E+) is formed as a major product of hydroethidine or dihydroethidium (HE or DHE) reaction with superoxide (O2•–) in intact animals with low tissue oxygen levels (J Cereb Blood Flow Metab 32:23–32, 2012). The authors concluded that measurement of ethidium (E+) is an indicator of O2•– formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems (Zhao et al, Free Radic Biol Med 34:1359, 2003 and Dikalov et al, Hypertension 49:717, 2007) showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O2•– and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O2•–. Results from these experiments further reaffirm our earlier findings (Zhao et al, Free Radic Biol Med 34:1359, 2003). We conclude that whether in vitro or in vivo, E+ measured by HPLC or by fluorescence lifetime imaging, is not a diagnostic marker product for O2•– reaction with HE.
fluorescence lifetime; hydroethidine; dihydroethidium; 2-hydroxyethidium; ethidium
F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the none-nzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.
Methods; Lipid oxidation; Isoprostanes; Oxidative stress; Mass spectrometry
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes which encode detoxifying enzymes, drug transporters (MRPs), anti-apoptotic proteins and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase mediated degradation of Nrf2. Chemicals including antioxidants, tocopherols including α-tocopherol (vitamin E), phytochemicals and radiations antagonize the Nrf2:INrf2 interaction and leads to the stabilization and activation of Nrf2. The signaling events involve pre-induction, induction and post-induction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinase(s) in a pre-induction response which phosphorylates specific residues on Nrf2 negative-regulators, INrf2, Fyn and Bach1, leading to their nuclear export, ubiquitination and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2cysteine151 followed by PKC phosphorylation of Nrf2serine40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus resulting in a coordinated activation of gene expression. This is followed by a post-induction response that controls the ‘switching off’ of Nrf2-activated gene expression. GSK3β under the control of AKT and PI3K, phosphorylates Fyn leading to Fyn nuclear localization. Fyn phosphorylates Nrf2Y568 resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provides protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
Nrf2; INrf2(Keap1); Antioxidants; Vitamins; Phytochemicals; ROS; Signaling; Regulation; Chemoprotection; Oncogenesis
Evolution has favored the utilization of dioxygen (O2) in the development of complex multi-cellular organisms. O2 is actually a toxic mutagenic gas that is highly oxidizing and combustible. It is thought that plants are largely to blame for polluting the earth’s atmosphere with O2 due to the development of photosynthesis by blue green algae over 2 billion years ago. The rise of the plants and atmospheric O2 levels placed evolutionary stress on organisms to adapt or become extinct. This implies that all the surviving creatures on our planet are mutants that have adapted to the “abnormal biology of O2.” Much of the adaptation to the presence of O2 in biological systems comes from well coordinated antioxidant and repair systems that focus on converting O2 to its most reduced form, water (H2O) and the repair and replacement of damaged cellular macromolecules. Biological systems have also harnessed O2’s reactive properties for energy production, xenobiotic metabolism, host defense, and as a signaling messenger and redox modulator of a number of cell signaling pathways. Many of these systems involve electron transport systems and offer many different mechanisms by which antioxidant therapeutics can alternatively produce an antioxidant effect without directly scavenging oxygen-derived reactive species. It is likely that each agent will have a different set of mechanisms that may change depending of the model of oxidative stress, organ system, or disease state. An important point is that all biological processes of aerobes have co-evolved with O2 and this creates a Pandora’s Box for trying to understand the mechanism of action(s) of antioxidants being developed as therapeutic agents.
We present arguments for an evolution in our understanding of how antioxidants in fruits and vegetables exert their health-protective effects. There is much epidemiological evidence for disease prevention by dietary antioxidants and chemical evidence that such compounds react in one-electron reactions with free radicals in vitro. Nonetheless, kinetic constraints indicate that in vivo scavenging of radicals is ineffective in antioxidant defense. Instead, enzymatic removal of non-radical electrophiles, such as hydroperoxides, in two-electron redox reactions is the major antioxidant mechanism. Furthermore, we propose that a major mechanism of action for nutritional antioxidants is the paradoxical oxidative activation of the Nrf2 (NF-E2-related factor 2) signaling pathway, which maintains protective oxidoreductases and their nucleophilic substrates. This maintenance of ‘Nucleophilic Tone,’ by a mechanism that can be called ‘Para-Hormesis,’ provides a means for regulating physiological non-toxic concentrations of the non-radical oxidant electrophiles that boost antioxidant enzymes, and damage removal and repair systems (for proteins, lipids, and DNA), at the optimal levels consistent with good health.
Oxidative stress is observed during aging and in numerous age-related diseases. Dietary restriction (DR) is a regimen that protects against disease and extends lifespan in multiple species. However, it is unknown how DR mediates its protective effects. One prominent and consistent effect of DR in a number of systems is the ability to reduce oxidative stress and damage. The purpose of this review is to comprehensively examine the hypothesis that dietary restriction reduces oxidative stress in rodents by decreasing reactive oxygen species (ROS) production and increasing antioxidant enzyme activity, leading to an overall reduction of oxidative damage to macromolecules. The literature reveals that the effects of DR on oxidative stress are complex and likely influenced by a variety of factors, including sex, species, tissue examined, types of ROS and antioxidant enzymes examined, and duration of DR. Here we present a comprehensive review of the existing literature on the effect of DR on mitochondrial ROS generation, antioxidant enzymes and oxidative damage. In a majority of studies, dietary restriction had little effect on mitochondrial ROS production or antioxidant activity. On the other hand, DR decreased oxidative damage in the majority of cases. Although the effects of DR on endogenous antioxidants are mixed, we find that glutathione levels are the most likely antioxidant to be increased by dietary restriction, which supports the emerging redox-stress hypothesis of aging.
Dietary restriction; calorie restriction; oxidative stress; reactive oxygen species; antioxidant enzymes; oxidative damage; aging
α-Tocopherol is a lipid-soluble antioxidant that is specifically required for reproduction and embryogenesis. However, since its discovery, α-tocopherol’s specific biologic functions, other than as an antioxidant, and the mechanism(s) mediating its requirement for embryogenesis, remain unknown. As an antioxidant, α-tocopherol protects polyunsaturated fatty acids (PUFAs) from lipid peroxidation. α-Tocopherol is likely required during embryonic development to protect PUFAs that are crucial to development, specifically arachidonic (ARA) and docosahexaenoic (DHA) acids. Additionally, ARA and DHA are metabolized to bioactive lipid mediators via lipoxygenase enzymes and α-tocopherol may directly protect, or it may mediate the production and/or actions of these lipid mediators. In this review, we discuss how α-tocopherol 1) prevents the nonspecific, radical-mediated peroxidation of PUFAs, 2) functions within a greater antioxidant network to modulate the production and/or function of lipid mediators derived from 12- and 12/15-lipoxygenase and 3) modulates 5-lipoxygenase activity. The application and implication of such interactions with be discussed in the context α-tocopherol requirements during embryogenesis.
7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, since it can be obtained non-invasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present when compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC-SRM/MS) with [15N5]-8-oxo-dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [13C1015N5]-dGuo and monitoring its conversion to [13C1015N5]-8-oxo-dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for inter-individual differences in urine flow by analysis of creatinine using stable isotope dilution LC-SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers when compared with non-smokers using either simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65 ng/mL and the levels normalized to creatinine were 1.72 μg/g creatinine. Therefore, stable isotope dilution LC-SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, where reduction in arachidonic acid precursor could confound isoP measurements.
stable isotope dilution; liquid chromatography-mass spectrometry; 8-oxo-dGuo; urine; oxidative DNA damage