Acute Oxidative Insult Research Articles

Transgenic Mouse Overexpressing Spermine Oxidase in Cerebrocortical Neurons: Astrocyte Dysfunction and Susceptibility to Epileptic Seizures.

Polyamines are organic polycations ubiquitously present in living cells. Polyamines are involved in many cellular processes, and their content in mammalian cells is tightly controlled. Among their function, these molecules modulate the activity of several ion channels. Spermine oxidase, specifically oxidized spermine, is a neuromodulator of several types of ion channel and ionotropic glutamate receptors, and its deregulated activity has been linked to several brain pathologies, including epilepsy. The Dach-SMOX mouse line was generated using a Cre/loxP-based recombination approach to study the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain. This mouse genetic model overexpresses spermine oxidase in the neocortex and is a chronic model of excitotoxic/oxidative injury and neuron vulnerability to oxidative stress and excitotoxic, since its phenotype revealed to be more susceptible to different acute oxidative insults. In this review, the molecular mechanisms underlined the Dach-SMOX phenotype, linked to reactive astrocytosis, neuron loss, chronic oxidative and excitotoxic stress, and susceptibility to seizures have been discussed in detail. The Dach-SMOX mouse model overexpressing SMOX may help in shedding lights on the susceptibility to epileptic seizures, possibly helping to understand the mechanisms underlying epileptogenesis in vulnerable individuals and contributing to provide new molecular mechanism targets to search for novel antiepileptic drugs.

Nogo-A aggravates oxidative damage in oligodendrocytes.

Nogo-A is considered one of the most important inhibitors of myelin-associated axonal regeneration in the central nervous system. It is mainly expressed by oligodendrocytes. Although previous studies have found regulatory roles for Nogo-A in neurite outgrowth inhibition, neuronal homeostasis, precursor migration, plasticity, and neurodegeneration, its functions in the process of oxidative injury are largely uncharacterized. In this study, oligodendrocytes were extracted from the cerebral cortex of newborn Sprague-Dawley rats. We used hydrogen peroxide (H2O2) to induce an in vitro oligodendrocyte oxidative damage model and found that endogenously expressed Nogo-A is significantly upregulated in oligodendrocytes. After recombinant virus Ad-ZsGreen-rat Nogo-A infection of oligodendrocytes, Nogo-A expression was increased, and the infected oligodendrocytes were more susceptible to acute oxidative insults and exhibited a markedly elevated rate of cell death. Furthermore, knockdown of Nogo-A expression in oligodendrocytes by Ad-ZsGreen-shRNA-Nogo-A almost completely protected against oxidative stress induced by exogenous H2O2. Intervention with a Nogo-66 antibody, a LINGO1 blocker, or Y27632, an inhibitor in the Nogo-66-NgR/p75/LINGO-1-RhoA-ROCK pathway, did not affect the death of oligodendrocytes. Ad-ZsGreen-shRNA-Nogo-A also increased the levels of phosphorylated extracellular signal-regulated kinase 1/2 and inhibited BCL2 expression in oligodendrocytes. In conclusion, Nogo-A aggravated reactive oxygen species damage in oligodendrocytes, and phosphorylated extracellular signal-regulated kinase 1/2 and BCL2 might be involved in this process. This study was approved by the Ethics Committee of Peking University People's Hospital, China (approval No. 2018PHC081) on December 18, 2018.

Increased basal antioxidant levels in RCAN1 – deficient mice lowers oxidative injury after acute paraquat insult

RCAN1 is an inhibitor of the phosphatase calcineurin, which is involved in the regulation of oxidative stress and apoptosis, among other important cell processes. Here we have used RCAN1 deficient mice (RCAN1-/-) to elucidate its role after an acute oxidative insult such as paraquat injection. We have observed that RCAN1-/- mice show less oxidative damage than wildtype (WT) mice after treatment. Under basal conditions, RCAN1-/- animals express more calcineurin, heme oxygenase-1, Nrf2, and catalase compared to WT mice (controls). This may explain the less severe effect of paraquat treatment on RCAN1-/- mice compared to WT. We showed that oxidative stress is involved in the early stages of apoptosis, thus we determined the apoptotic effector BAD and found that decreases in RCAN1-/- mice after treatment with paraquat compared with WT in similar experimental conditions. Our results suggest that RCAN1 may be involved in the balance between oxidant and antioxidant species production in vivo.

Protective effects of tiopronin on oxidatively challenged human lung carcinoma cells (A549)

Tiopronin (MPG) is a thiol antioxidant drug that has been explored as a treatment for various oxidative stress-related disorders. However, many of its antioxidant capabilities remain untested in well-validated cell models. To more thoroughly understand the action of this promising pharmaceutical compound against acute oxidative challenge, A549 human lung carcinoma cells were exposed to tert-butyl hydroperoxide (tBHP) and treated with MPG. Analyses of cell viability, intracellular glutathione (GSH) levels, and the prevalence of reactive oxygen species (ROS) and mitochondrial superoxide were used to examine the effects of MPG on tBHP-challenged cells. MPG treatment suppressed intracellular ROS and mitochondrial superoxide and prevented tBHP-induced GSH depletion and apoptosis. These results indicate that MPG is effective at preserving redox homeostasis against acute oxidative insult in A549 cells if present at sufficient concentrations during exposure to oxidants such as tBHP. The effects of treatment gleaned from this study can inform experimental design for future in vivo work on the therapeutic potential of MPG.

Abstract 279: Effects of Acute Oxidative Stress on Cardiac Proteome Dynamics

Introduction: Acute reactive oxygen species exposure is thought to trigger endoplasmic reticulum (ER) stress and unfolded protein response (UPR) commonly observed in pathological hearts. The pathways and molecular features impacted by acute oxidative stress are incompletely understood. To understand how proteostatic stressors impact cardiac protein dynamics, we investigate the effect of low to high doses of acute oxidant on the cardiac proteome using a paraquat-induced oxidative stress model. Methods: C57BL/6J mice were exposed to low, mid, or high doses of paraquat for up to 48 hours. Cardiac proteins were extracted and digested on-filters into tryptic peptides. The peptides were then tagged with isobaric stable isotope tandem mass tags (TMT) followed by high-pH reversed-phase (RP) fractionation. Each fraction was further separated by low-pH RP liquid chromatography and analyzed on a Q-Exactive HF Plus high-resolution mass spectrometer to obtain proteome-wide abundance levels across treatment groups. We then focused on the differential expression in the mitochondrial and ER subproteomes as well as their respective protein quality control pathways. Results: We found prominent differential expression in specific quality control proteins in response across low and high doses of oxidative stresses. The data suggest that oxidative stress activates UPR pathways including notably mitochondrial UPR (UPRmt) components in a dose-specific manner. In particular, we observed the repression of mitochondrial protein import machinery components as a specific feature of low but not high oxidative stress response. In addition, the differential expression of pyruvate metabolism proteins may constitute a link between cardiac metabolism and UPRmt, and serve as a determinant between adaptive and maladaptive responses to reactive oxygen species. Conclusion: Our results indicate that the activation of the UPRmt pathways may be an overlooked feature of acute oxidative insults that underlies stress response in cardiac cells. Future work will interrogate the molecular crosstalks between mitochondria and ER stress response pathways.

Cocoa ingestion protects plasma lipids in healthy males against ex vivo oxidative conditions: A randomized clinical trial

The effects of invivo cocoa-based supplementation were studied as a preconditioning treatment for exvivo acute oxidative conditions in a controlled randomized clinical trial. Subjects were 100 healthy young men at Universidad Industrial de Santander blinded to the intervention and divided into two groups: The intervention group (n=50) receiving 30g of cocoa powder and 50g of dark chocolate/d for 1 week with the remaining subjects receiving placebo. Cocoa products preconditioning for 1 week resulted in modifications in the susceptibility of plasma lipids over exvivo oxidative conditions with effects of i) a significant increase in the oxidative resistance of plasma lipids measured by dienes formation (4.2, CI: 0.18, 8.2; P=0.04), and ii) a significant reduction in the production of toxic aldehydes as established by a decrease in the content of hexanal, quantified by gas chromatography (-0.22, CI:-0.38,-0.05; P=0.009). The invivo cocoa-based preconditioning demonstrated protective properties against exvivo oxidative modifications, improving total plasma lipids resistance to oxidation and protecting plasma lipids against great acute oxidative insult in comparison with placebo. This trial was registered at clinical clinicaltrials.gov as NCT01347450.

100 - Mechanism of Neuroprotection by Capsaicin in a Model of Parkinson's Disease

Parkinson’s disease (PD) is characterised by progressive loss of motor function and dopaminergic neuron (DN) density. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is neurotoxic to DN in the mouse substantia nigra yielding PD-like symptoms. Red peppers contain capsaicin that can protect neurons in an in vitro model of acute oxidative insult (Fong and Witting unpublished). We aim to test whether capsaicin prevents neuronal loss and brain function in a mouse model of PD. Male C57BL/6 mice were pre-treated with vehicle or capsaicin (1 mg/kg) prior to saline or MPTP (25 mg/kg/day) treatment over 2 consecutive days. Gait was assessed on a subset of mice six days after the onset of treatment. Serum and various organs were harvested for biochemical and histological analyses. MPTP-treated mice displayed signs of akinesia and increased number of abnormal steps; this was restored to normal with capsaicin pre-treatment. Total DN counts decreased in the MPTP-treated animals while capsaicin pre-treatment improved viability. Cerebral levels of IL-6, TNF-α and IFN-γ remained unchanged. MPTP treatment increased SOD and decreased catalase activity while capsaicin pre-treatment restored SOD and catalase activity to control levels. The dose of MPTP employed did not significantly alter gait dynamics but as anticipated did induce noticeable decline in DN density. Capsaicin pre-treatment protected DN from MPTP, possibly via an augmented antioxidant system.

Abstract 2919: Different ROS control mechanisms and mutagenic consequences in primitive subsets of normal human mammary cells.

Abstract Reactive oxygen species (ROS) are known mediators of DNA damage and likely contributors to oncogenesis. However, very little is known about the mechanisms controlling ROS in the cells that make up the normal human mammary gland, in spite of its being a major site of cancer development. To investigate how ROS are generated and their potential role in two functionally distinct, primitive normal human mammary epithelial cell compartments (luminal and basal), we isolated these subsets at high purities by FACS and compared the levels within them of ROS, components that positively and negatively regulate ROS, their responses to oxidative stressors and evidence of ROS-associated DNA damage. The results show that purified progenitors of the cells that line the gland lumen (defined by their EpCAM+ CD49f+ phenotype) contain significantly higher levels of superoxide (O2*) anions and H2O2 than the basal cells (defined as EpCAMlow/- CD49fhigh cells and highly enriched in bipotent progenitors and stem cells). We also find that the elevated levels of these ROS elements in the luminal progenitors are associated with a higher content of mitochondria and higher levels of all 3 superoxide dismutases (SOD-1, 2 and 3). The luminal progenitors are also highly resistant to glutathione depletion and express higher levels of both non-canonical non-glutathione anti-oxidant enzymes and multiple enzymes that control ROS-induced nucleotide damage (i.e., OGG-1, MTH-1, MUTYH) providing a likely explanation for their ability to survive following GSH depletion. Interestingly, we found the mitochondrial antioxidant glutathione peroxidase (GPX)-2 enzyme to be expressed almost exclusively and at high levels in basal mammary cells and its depletion, using a shRNA lentivirus, resulted in loss of progenitor viability/activity by basal but not luminal cells. Luminal progenitors also displayed greater resistance to acute oxidative insults (H2O2 and X-radiation) and displayed an increased accrual of oxidative damage-induced (genomic 8-oxo-dGTP) mutations. Our findings reveal a major difference in the molecular machinery that control ROS levels in normal human basal and luminal mammary progenitors. These correlate with an ability of the luminal progenitors to maintain and tolerate elevated levels of ROS and the continuous acquisition of unrepaired ROS-induced DNA damage, thus suggesting a novel and previously unanticipated vulnerability of these cells to undergo oncogenic transformation. (Supported by Canadian Breast Cancer Foundation/BC-Yukon and the Canadian Cancer Society) Citation Format: Nagarajan Kannan, Maisam Makarem, Long Nguyen, Jeff Dong, Peter Eirew, Connie Eaves. Different ROS control mechanisms and mutagenic consequences in primitive subsets of normal human mammary cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2919. doi:10.1158/1538-7445.AM2013-2919

Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells

In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2), despite the fact that physiological O2 at the tissue level in vivo is much lower (~1–5% O2). We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation) or oxidative damage to proteins (protein disulfides, carbonyls and aggregates) in myoblast precursor cells (MPCs) isolated from young (3–4m) and old (29–30m) C57BL/6 mice. MPCs were grown under physiological (3%) or ambient (21%) O2 for two weeks prior to exposure to an acute oxidative insult (H2O2). Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

Impaired adaptability of in vivo mitochondrial energetics to acute oxidative insult in aged skeletal muscle

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires 31P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O2 concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration.

Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H(2)O(2)). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H(2)O(2) treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure-function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290-562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.

Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow

Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously. Subsequently, the response of a redox homeostasis circuit was tested, namely reduced glutathione (GSH)/oxidized glutathione(GSSG), to diamide, a thiol oxidant, and two drugs used for cancer therapies: BSO (L-buthionine-[SR]-sulfoximine) and BCNU (carmustine). The main outcome from these experiments is a comparison of the temporal depletion and recovery of GSH in single living cells in real-time. These data demonstrate that mammalian cells are capable of restoring a reduced intracellular redox environment in minutes after an acute oxidative insult is removed. This recovery is significantly delayed by (i) the inhibition of GSH biosynthesis by BSO; (ii) the inactivation of glutathione reductase by BCNU; and (iii) in tumorigenic cells relative to an isogenic non-tumorigenic control cell line.

Oxidative stress in NPC1 deficient cells: protective effect of allopregnanolone

Niemann-Pick C disease (NPC) is an autosomal recessive neurodegenerative disorder caused by the abnormal function of NPC1 or NPC2 proteins, leading to an accumulation of unesterified cholesterol and glycosphingolipids (GSLs) in the lysosomes. The mechanisms underlying the pathophysiology in NPC disease are not clear. Oxidative damage is implicated in the pathophysiology of different neurological disorders and the effect of GSL accumulation on the intracellular redox state has been documented. Therefore, we determined whether the intracellular redox state might contribute to the NPC disease pathophysiology. Because the treatment of NPC mice with allopregnanolone (ALLO) increases their lifespan and delays the onset of neurological impairment, we analysed the effect of ALLO on the oxidative damage in human NPC fibroblasts. Concentrations of reactive oxygen species (ROS) and lipid peroxidation were higher in fibroblasts from NPC patients than in fibroblasts from normal subjects. Fibroblasts from NPC patients were more susceptible to cell death through apoptosis after an acute oxidative insult. This process is mediated by activation of the NF-κB signalling pathway. Knockdown of NPC1 mRNA both in normal fibroblasts and in human SH-SY5Y neuroblastoma cells caused increased ROS concentrations. ALLO treatment of fibroblasts from NPC patients or NPC1 knockdown cells reduced the levels of ROS and lipid peroxidation and prevented peroxide-induced apoptosis and NF-kB activation. Thus, these findings suggest that oxidative stress might contribute to the NPC disease and ALLO might be beneficial in the treatment of the disease, at least in part, due to its ability to restore the intracellular redox state.

Overexpression of Peroxiredoxin 6 Does Not Prevent Ethanol-Mediated Oxidative Stress and May Play a Role in Hepatic Lipid Accumulation

Oxidative stress is implicated in the etiology of many diseases, including alcoholic liver disease (ALD). Peroxiredoxin 6 is a cytosolic peroxidase that has been demonstrated to protect various tissues, such as skin, lung, and cardiac muscle, against acute oxidative insults. Consequently, peroxiredoxin 6 was hypothesized to also protect the liver from oxidative stress generated during the process of chronic ethanol ingestion. To test this, wild-type peroxiredoxin 6 knockout mice (KO), and transgenic peroxiredoxin 6 overexpressing mice (TG) were fed an ethanol-containing diet. Various biomarkers of ALD were assessed, along with the effects of chronic ethanol consumption on the antioxidant defenses. After 9 weeks of ethanol consumption, all backgrounds exhibited elevations of plasma alanine aminotransferase activity, hepatosteatosis, CYP2E1 induction, and lipid peroxidation; however, hepatic triglyceride accumulation seemed to be exacerbated in ethanol-fed TG mice. Differences in antioxidant protein expression and activity in response to chronic ethanol consumption were also observed. Examples include significant inductions of catalase and glutathione transferase activity in ethanol-fed KO and TG mice, along with elevated levels of glutathione peroxidase activity. These alterations in antioxidant defenses could be attributed to either compensatory responses due to the genetic manipulations or ethanol-mediated responses. In conclusion, both ethanol-fed KO and ethanol-fed TG mice developed early stage ALD and peroxiredoxin 6 may play a role in ethanol-mediated hepatic lipid accumulation.

Role of wild-type estrogen receptor-β in mitochondrial cytoprotection of cultured normal male and female human lens epithelial cells

The influence of sexual category as a modifier of cellular function is underinvestigated. Whether sex differences affect estrogen-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from postmortem male and female donors. Experimental indicators assessed included differences in estrogen receptor-beta (ERbeta) isoform expression, receptor localization in mitochondria, and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound JC-1 after nHLE were exposed to peroxide. The impact of wild-type ERbeta (wtERbeta1) was also assessed using wtERbeta1 siRNA to suppress expression. A triple-primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtERbeta1, -beta2, and -beta5) from the total ERbeta message pool in male and female cell cultures. Irrespective of sex, nHLE express wtERbeta1 and the ERbeta2 and ERbeta5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria as well as nuclear staining in both cell populations. The ERbeta2 and ERbeta5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with E(2) (1 muM) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtERbeta1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of E(2). Thus, whereas the estrogen-mediated prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtERbeta1 dependent.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H 2O 2) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H 2O 2 treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor αplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.