Basal Levels Of Oxidative Stress Research Articles

Role of inorganic nanoparticle degradation in cancer therapy.

Nanomaterials are currently widely exploited for their potential in the development of novel cancer therapies, and so far, mainly nanoparticles (NPs) consisting of liposomes and polymers have made their way into the clinic. However, major bottlenecks for the clinical translation of other types of NPs (i.e. inorganic) are the lack of knowledge concerning their long-term distribution in vivo and their potential toxicity. To counter this, various research groups have worked on soluble NPs, such as zinc oxide (ZnO), copper oxide (CuO), and silver (Ag), which tend to dissolve spontaneously into their ionic form, releasing toxic metal ions and leading to reactive oxygen species (ROS) generation when exposed to cellular environments. By fine-tuning the dissolution kinetics of these NPs, it is possible to control the level of ROS production and thus cytotoxicity to selectively destroy tumor tissue. Specifically, cancer cells tend to exhibit a higher basal level of oxidative stress compared to normal cells due to their higher metabolic rates, and therefore, by engineering NPs that generate sufficient ROS that barely exceed toxic thresholds in cancer cells, normal cells will only experience reversible transient damage. This review focuses on the use of these soluble inorganic NPs for selective cancer therapy and on the various in vitro and in vivo studies that have aimed to control the dissolution kinetics of these NPs, either through particle doping or surface modifications.

Paternal impacts on development: identification of genomic regions vulnerable to oxidative DNA damage in human spermatozoa.

Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. Oxidative DNA damage is frequently encountered in the spermatozoa of male infertility patients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactive oxygen species. Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent samples and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). Human spermatozoa were obtained from normozoospermic males and divided into two identical samples prior to being incubated with either H2O2 (5mm, 1h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen samples were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. Approximately 9000 genomic regions, 150-1000bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2-15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. The limited number of samples analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. Project support was provided by the University of Newcastle's (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.

324 - Investigation of cytoplasmic and mitochondrial redox status in Down syndrome: impact of the thioredoxin and glutathione systems

Down syndrome (DS) results from the triplication of whole or part of chromosome 21. Individuals with DS experience a variety of comorbidities at a higher rate than the euploid population, and many of these pathologies involve oxidative stress as an etiological factor. Blood and postmortem tissue samples from DS individuals indicate elevated basal levels of oxidative stress, potentially due to mitochondrial dysfunction. Due to these observations we hypothesized that cells from DS individuals would display greater basal oxidation in the cytoplasmic and mitochondrial compartments, and that manipulation of the thioredoxin (Trx) or glutathione (GSH) systems would exacerbate this oxidation. To investigate this, we employed four pairs of patient-derived euploid or DS fibroblasts, and utilized auranofin or BSO to selectively modulate the Trx and GSH systems, respectively. Additionally, peroxiredoxin (Prx) redox Western blots were used to assess cytoplasmic (Prx1) and mitochondrial (Prx3) redox status. Results from these studies showed that inhibition of the Trx system via auranofin induced significant oxidation of both cytoplasmic and mitochondrial compartments. Interestingly, depletion of GSH by BSO did not cause significant oxidation of either Prx1 or Prx3 in DS vs. euploid controls. Mechanistically, increased sensitivity in compartmental oxidation observed in DS may be explained by our observation of significantly decreased protein levels of thioredoxin reductase (TrxR) 1 and 2, as well as significantly decreased TrxR activity in DS. Together, these data indicate that DS individuals may be highly susceptible to exposures or treatments known to oxidize the thiol redox proteome, thereby enhancing the risk of initiation and progression of DS-related comorbidities.

Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells

BackgroundRecent studies have shown that low density lipoproteins reconstituted with the natural omega 3 fatty acid docosahexaenoic acid (LDL-DHA) is selectively cytotoxic to liver cancer cells over normal hepatocytes. To date, little is known about the subcellular events which transpire following LDL-DHA treatment. MethodsHerein, murine noncancer and cancer liver cells, TIB-73 and TIB-75 respectively, were investigated utilizing confocal microscopy, flow cytometry and viability assays to demonstrate differential actions of LDL-DHA nanoparticles in normal versus malignant cells. ResultsOur studies first showed that basal levels of oxidative stress are significantly higher in the malignant TIB-75 cells compared to the normal TIB-73 cells. As such, upon entry of LDL-DHA into the malignant TIB-75 cells, DHA is rapidly oxidized precipitating global and lysosomal lipid peroxidation along with increased lysosomal permeability. This leakage of lysosomal contents and lipid peroxidation products trigger subsequent mitochondrial dysfunction and nuclear injury. The cascade of LDL-DHA mediated lipid peroxidation and organelle damage was partially reversed by the administration of the antioxidant, N-acetylcysteine, or the iron-chelator, deferoxamine. LDL-DHA treatment in the normal TIB-73 cells was well tolerated and did not elicit any cell or organelle injury. ConclusionThese studies have shown that LDL-DHA is selectively cytotoxic to liver cancer cells and that increased levels of ROS and iron catalyzed reactions promote the peroxidation of DHA which lead to organelle dysfunction and ultimately the demise of the cancer cell. General significanceLDL-DHA selectively disrupts lysosomal, mitochondrial and nuclear function in cancer cells as a novel pathway for eliminating cancer cells.

Targeting Redox Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Proteasome inhibitors (PIs) like bortezomib (Btz) and carfilzomib (Crflz) induce an oxidative stress response in Multiple Myeloma (MM) cells. Oxidative stress is a key effector pathway in PI-induced cell death, and altered redox signaling has been implicated in the acquisition of PI resistance. The potential of redox as a therapeutic target/pathway for PI resistant MM has not been realized due to the absence of a precise molecular targeted strategy that exploits redox signaling in a way that attacks PI resistant cells while sparing normal cells. Therefore, we set out in this study to characterize redox adaptations that contribute to PI resistance in MM, and to use drug screening platforms to identify specific redox-targeted small molecules that restore PI sensitivity. Using multiple isogenic pairs of PI sensitive and resistant MM cell lines, we found that resistant cells exist under high basal levels of reactive oxygen species (ROS) and oxidation of protein thiols (i.e., oxidative damage). Resistant cells induce significantly higher relative levels of ROS following PI treatment, but exhibit no further increase in oxidative damage. By comparison, their PI sensitive counterparts have relatively low levels of basal and PI-induced ROS levels, but undergo significantly higher levels of oxidative damage following PI treatment. These findings demonstrate that PI resistance is associated with alterations in redox balance; they further suggest that PI resistant cells have acquired adaptations that allow them to survive under high basal levels of oxidative stress, and that provide protection from PI-induced oxidative damage. We also identified significant changes in cellular bioenergetics that are typical of PI resistant cells. Generally, PI resistant cells appear to be more metabolically efficient, relying on mitochondrial respiration as their primary source of ATP production. Specifically, PI resistant cells have higher basal oxygen consumption rates (OCR), expanded respiratory capacity, increased NAD(P)H levels and pyruvate dehydrogenase (PDH) activity, and nearly absent activation of the AMP kinase energy stress signaling pathway. Thus, the acquisition of PI resistance is associated with significant changes in redox balance as well as in cellular bioenergetics. Given these findings, we next used a cell-based drug screening method to screen for redox-targeted small molecules capable of restoring PI sensitivity to resistant cells. We screened a compound collection of known pro- and anti-oxidant small molecules with wide-ranging mechanisms of action. From this screen we identified compound E61, which demonstrated strong synergy with multiple PIs, including Btz, Crflz, ixazomib, and oprozomib. E61 induced an oxidative stress response characterized by a burst of ROS generation and oxidation of protein thiols, and synergistically enhanced the PI-induced oxidative stress response in resistant cells. The synergistic cytotoxic response to E61 and PI co-treatment was dependent on ROS, and was evident across several models of PI resistance, representing cells of diverse genetic backgrounds. While E61 enhanced PI-induced cell death in resistant MM cells, its effects were protective in normal cell types, including peripheral blood mononuclear cells (PMBCs) and lymphocytes from normal human donors. These findings suggest that compound E61 will have a wide therapeutic index in combination with PI therapy in preclinical mouse models of MM, a hypothesis that we are currently testing. All together, our findings identify specific redox and bioenergetics changes that are acquired by PI resistant MM cells. Furthermore, our work offers a novel redox-targeted small molecule, E61, to be used in combination with PI-based therapeutic regimens in refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Type II muscle is more susceptible to contractile activity‐induced injury

Contractile activity (CA) induces changes of cellular environment in cells, such as increased oxidative stress or inflammation. The changes are double‐ edged sword. When they are subtle and transient, they serve as signals of muscle growth induced by CA. However, cells could be damaged if CA‐induced oxidative stress and inflammation are excessive and prolonged. To date, it is unknown whether the responses to aerobic CA are fiber‐type specific. The aim of the study is to determine the effects of skeletal muscle types on inflammation and oxidative stress following aerobic CA. Type I (soleus) and Type II (white gastrocnemius) muscles from stimulated and contralateral non‐stimulated limbs were collected immediately and 6 hours after 21‐mintues of aerobic CA. Markers of oxidative stress (GSH, GSH/GSSG, oxidative‐modified proteins and lipids), inflammation (TNFα, IL6), growth factors (IGF1, MGF and myostatin), myogenesis (Myf5), and protein degradation (MuRF1) were evaluated. Independent t test was used to compare parameters between groups. Compared to type I muscles, type II muscles had lower basal level of oxidative stress (less accumulation of oxidative‐modified proteins and lipids, lower GSH level and GSH/GSSG), lower inflammation state (lower TNFα and IL6 levels), and lower levels of signals that stimulate the increase of muscle mass (lower levels of IGF1, MGF and Myf5, and higher level of myostatin and MuRF1). In response to aerobic CA, type II muscles showed earlier accumulation of oxidative damages than that in type I muscles. In addition, the Myf5 level in type I muscles elevated at 6 h after CA, but this change was not observed in type II muscles. Type II muscle is more susceptible to contractile activity‐induced injury. In addition, the regeneration capacity of type II muscle following aerobic CA is weaker than type I muscle.

Increased oxidative stress with reduced responsiveness of circulating leukocytes following pulmonary metal‐rich particulate matter exposure

Pulmonary exposure to metal‐rich particulate matter (PM) generated from welding causes localized immunosuppression. The aim of this study was to evaluate the molecular changes and responsiveness of circulating leukocytes following exposure. Rats were exposed to manual metal arc stainless steel welding fume (MMAW‐SS) at 2 mg/rat by intratracheal instillation and sacrificed at 4 and 24 hr post‐exposure. Collected whole blood was then challenged with and without LPS for 24 hr. After incubation, supernatants were collected for protein analysis and the cellular fraction was collected for global gene expression changes. Also, mononuclear cells were isolated and analyzed for oxidative stress by flow cytometry and confocal microscopy. Ex vivo leukocyte stimulation with LPS showed reduced production of CCL4, CXCL2, CXCL10 and TNF‐alpha protein in MMAW‐SS treated rats. Interestingly, cellular gene expression changes from MMAW‐SS and PBS rats were similar indicating effects were not related to transcription. Since oxidative stress can affect translation, isolated mononuclear cells were tested and showed increased basal levels of oxidative stress. These results showed increased oxidative stress with a reduced responsiveness of circulating leukocytes providing mechanistic insight into epidemiological and experimental evidence illustrating immunosuppression following a metal‐rich PM exposure.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Tipping the Balance in the Powerhouse of the Cell to “Protect” Colorectal Cancer

Tipping the Balance in the Powerhouse of the Cell to “Protect” Colorectal Cancer

High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis

Reactive oxygen species (ROS) are strongly implicated in melanoma development, and treatment with antioxidants has shown efficacy in suppressing malignant transition and progression. Here, we investigated the significance of the glutamate-L: -cysteine ligase catalytic subunit (GCLC) expression, a key regulator of glutathione synthesis, for malignant melanoma. A large set of melanoma cell lines (n = 36) was analyzed, and higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. Moreover, treatment with the antioxidant N-acetylcysteine efficiently reduced the growth speed of several investigated malignant cells. In addition GCLC expression was significantly linked to a prominent set of cellular antioxidants, accounting for the observed lower basal levels of oxidative stress and higher antioxidative capacity. Key attributes defining the malignant phenotype of melanoma cells including survival, invasiveness, and switch from E-cadherin to N-cadherin expression were more prominent in cells with lower GCLC expression. Our findings were further corroborated by observations in Rag2(-/-)γc(-/-)mice, in which melanoma cells with lower GCLC expression depicted a dramatically stronger tumor growth. Furthermore, prognostic significance of GCLC expression was investigated in patients (n = 28) with advanced malignant melanoma. High tumor immunoreactivity for GCLC was a significant determinant for better 5-year overall survival. Conclusively, we show for the first time that GCLC may serve a dual role, as a surrogate marker for cellular redox state as well as malignant potential of melanoma cells. These promising results regarding its prognostic significance as well as its potential as a pharmacological target require further in-depth investigations.

Oxidative stress in older adults: effects of physical fitness

Acute exercise results in transient change in redox balance. High concentrations of reactive oxygen species (ROS) can lead to oxidative damage to macromolecules. However, moderate periodic increases in ROS, such as experienced with habitual exercise, may activate signal transduction pathways which stimulate increases in endogenous antioxidant systems. This study tested the hypothesis that physically fit older adults would have less oxidative stress than unfit age-matched controls, due to greater circulating concentrations of non-enzymatic antioxidants and greater capacity to upregulate antioxidant enzymes. We compared 37 fit (mean age 65.2 ± 5years) and 35 unfit (mean age 67.7 ± 4years) men and women. Fitness status was classified by VO(2max) and maximal leg power. Basal levels of oxidative stress were assessed by measuring urinary markers of nucleic acid damage and lipid peroxidation. Antioxidant status was assessed by measuring total antioxidant power and ratios of reduced to oxidized glutathione in plasma, at rest. The capacity to counteract an oxidative insult was assessed by measuring changes in plasma F(2)-isoprostanes in response to forearm ischemia-reperfusion. The fit individuals had significantly lower levels of urinary markers of oxidative damage (all P <0.05) and lower F(2)-isoprostane response to the oxidative challenge (P < 0.05), but there were no group differences in antioxidant status. The lower levels of oxidative stress in the fit individuals were not mediated by known effects of exercise training such as adiposity, HDL concentrations, or small molecular weight antioxidants. These data suggest that reduced oxidative stress associated with physical fitness results from differences in activity of antioxidant enzymes.

AGE restriction in diabetes mellitus: a paradigm shift

Persistently elevated oxidative stress and inflammation precede or occur during the development of type 1 or type 2 diabetes mellitus and precipitate devastating complications. Given the rapidly increasing incidence of diabetes mellitus and obesity in the space of a few decades, new genetic mutations are unlikely to be the cause, instead pointing to environmental initiators. A hallmark of contemporary culture is a preference for thermally processed foods, replete with pro-oxidant advanced glycation endproducts (AGEs). These molecules are appetite-increasing and, thus, efficient enhancers of overnutrition (which promotes obesity) and oxidant overload (which promotes inflammation). Studies of genetic and nongenetic animal models of diabetes mellitus suggest that suppression of host defenses, under sustained pressure from food-derived AGEs, may potentially shift homeostasis towards a higher basal level of oxidative stress, inflammation and injury of both insulin-producing and insulin-responsive cells. This sequence promotes both types of diabetes mellitus. Reducing basal oxidative stress by AGE restriction in mice, without energy or nutrient change, reinstates host defenses, alleviates inflammation, prevents diabetes mellitus, vascular and renal complications and extends normal lifespan. Studies in healthy humans and in those with diabetes mellitus show that consumption of high amounts of food-related AGEs is a determinant of insulin resistance and inflammation and that AGE restriction improves both. This Review focuses on AGEs as novel initiators of oxidative stress that precedes, rather than results from, diabetes mellitus. Therapeutic gains from AGE restriction constitute a paradigm shift.

Inhibition of xanthine oxidase reduces oxidative stress and improves skeletal muscle function in response to electrically stimulated isometric contractions in aged mice

Oxidative stress is a putative factor responsible for reducing function and increasing apoptotic signaling in skeletal muscle with aging. This study examined the contribution and functional significance of the xanthine oxidase enzyme as a potential source of oxidant production in aged skeletal muscle during repetitive in situ electrically stimulated isometric contractions. Xanthine oxidase activity was inhibited in young adult and aged mice via a subcutaneously placed time-release (2.5mg/day) allopurinol pellet, 7days before the start of in situ electrically stimulated isometric contractions. Gastrocnemius muscles were electrically activated with 20 maximal contractions for 3 consecutive days. Xanthine oxidase activity was 65% greater in the gastrocnemius muscle of aged mice compared to young mice. Xanthine oxidase activity also increased after in situ electrically stimulated isometric contractions in muscles from both young (33%) and aged (28%) mice, relative to contralateral noncontracted muscles. Allopurinol attenuated the exercise-induced increase in oxidative stress, but it did not affect the elevated basal level of oxidative stress that was associated with aging. In addition, inhibition of xanthine oxidase activity decreased caspase-3 activity, but it had no effect on other markers of mitochondrial-associated apoptosis. Our results show that compared to control conditions, suppression of xanthine oxidase activity by allopurinol reduced xanthine oxidase activity, H2O2 levels, lipid peroxidation, and caspase-3 activity; prevented the in situ electrically stimulated isometric contraction-induced loss of glutathione; prevented the increase in catalase and copper–zinc superoxide dismutase activities; and increased maximal isometric force in the plantar flexor muscles of aged mice after repetitive electrically evoked contractions.

Reduced cyclic stretch, endothelial dysfunction, and oxidative stress: an ex vivo model

The objective of this study was to investigate whether reduction of cyclic circumferential stretch will impair endothelial function and elevate basal levels of oxidative stress, both known risk factors linked to cardiovascular disease. Ex vivo and in vitro models were used to perfuse porcine carotid arteries and porcine endothelial cells, respectively, for 24 h. In both cases, one group was allowed to stretch naturally when exposed to a pulse shear stress (6+/-3 dynes/cm(2)) combined with a pulse pressure of 80+/-10 mmHg, yielding a physiological cyclic stretch of 4-5%. This group was compared to a reduced stretch group, achieved by wrapping the arterial segment with a silicon band or by seeding the endothelial cells inside less compliant tubes, decreasing cyclic stretch to 1%. The experimentally reduced compliance caused a significant decrease in bradykinin-dependent vascular relaxation. Reduced compliance significantly decreased the phosphorylation of serine 1177 (Ser1177) on eNOS, suggesting the activity of eNOS was decreased. Overall production of reactive oxygen species was increased by reducing compliance, as visualized with DHE. Finally, p22-phox and p47-phox, key players in the superoxide-generating NAD(P)H oxidase, were also up-regulated by reduced compliance. These findings point out how reduced arterial compliance increases the risk of arterial disease by creating a less functional endothelium, interrupting the eNOS activation pathway, and increasing the vascular levels of oxidative stress.

Nitric Oxide Evokes an Adaptive Response to Oxidative Stress by Arresting Respiration

Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNgamma-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H(2)O(2). NADH antagonizes the hydroxyl radical (OH(.)) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.

Lipid Peroxidation During Human Cerebral Myelination

The critical period of human cerebral myelination is characterized by rapid production of cellular membranes. We hypothesize that this period is subject to the "physiological" generation of free radicals resulting in lipid peroxidation (LPO). In this study, oxidative markers were examined in developing human parietal white matter using 4-hydroxy-2-nonenal (HNE) protein adducts as an indicator of LPO. Immunocytochemistry showed an increase in HNE-positive glia from 40 gestational weeks to 1.5 postnatal years encompassing the peak period of myelin sheath synthesis at this site. Western blots showed a distinct pattern of HNE-modified proteins at fetal/term ages 26 to 42 gestational weeks and a second, different pattern at 45 gestational weeks to 2.5 postnatal years. Proteins modified by HNE in the latter period, corresponding to active myelination, were identified using mass spectrometry. The most prominent category of HNE modification included cytoskeletal proteins such as tubulins and neurofilaments. Other categories included cell type-specific proteins for mature oligodendrocytes and astrocytes and proteins involved in cell cycle and energy metabolism. We conclude that human brain development involves basal levels of oxidative stress and resulting LPO and that these processes target different proteins in an age-specific manner, thereby likely playing distinct roles during different periods of brain maturation.

Heme oxygenase-1 production by peripheral blood monocytes during acute inflammatory illnesses of children.

Monocytes play key roles both in innate and adaptive antigen-specific immunity and they constitute critical components of the immune responses. Although most of the monocyte-derived cytokines exhibit proinflammatory functions in vivo, heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, exerts potent anti-inflammatory effect through production of carbon monoxide and bilirubin. We compared HO-1 production by monocytes in vivo in various acute inflammatory illnesses and in normal controls. Freshly isolated monocytes produced little HO-1 as detected by immunohistochemistry, but it was rapidly induced in vitro upon stimulation. HO-1 production by monocytes was selective because it was not induced in other leukocyte populations, including granulocytes and lymphocytes. Monocytes from acute inflammatory illnesses, such as Kawasaki disease and acute infectious diseases, viral or bacterial, produced significant levels of HO-1, as detected by flow cytometry, immunohistochemistry, and reverse transcription polymerase chain reaction. Quantitative analysis of HO-1 mRNA expression by real-time polymerase chain reaction revealed that monocytes from controls exhibited low, but significant levels of HO-1 mRNA, indicating that circulating monocytes produce HO-1 constantly, in response to basal level of oxidative stress encountered daily. Significantly elevated HO-1 mRNA levels seen in acute inflammatory illnesses suggest that monocyte HO-1 production serve as potent anti-inflammatory agent to control excessive cell or tissue injury in the presence of oxidative stress and cytokinemia.

Escherichia coli aconitases and oxidative stress: post-transcriptional regulation of sodA expression.

Escherichia coli possesses two aconitases, a stationary-phase enzyme (AcnA), which is induced by iron and oxidative stress, and a major but less stable enzyme (AcnB), synthesized during exponential growth. In addition to the catalytic activities of the holo-proteins, the apo-proteins function as post-transcriptional regulators by site-specific binding to acn mRNAs. Thus, it has been suggested that inactivation of the enzymes could mediate a rapidly reacting post-transcriptional component of the bacterial oxidative stress response. Here it is shown that E. coli acn mutants are hypersensitive to the redox-stress reagents H(2)O(2) and methyl viologen. Proteomic analyses further revealed that the level of superoxide dismutase (SodA) is enhanced in acnB and acnAB mutants, and by exposure to methyl viologen. The amounts of other proteins, including thioredoxin reductase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase and chaperone proteins, were also affected in the acn mutants. The altered patterns of sodA expression were confirmed in studies with sodA-lacZ reporter strains. Quantitative Northern blotting indicated that AcnA enhances the stability of the sodA transcript, whereas AcnB lowers its stability. Direct evidence that the apo-proteins have positive (AcnA) and negative (AcnB) effects on SodA synthesis was obtained from in vitro transcription-translation experiments. It is suggested that the aconitase proteins of E. coli serve as a protective buffer against the basal level of oxidative stress that accompanies aerobic growth by acting as a sink for reactive oxygen species and by modulating translation of the sodA transcript.

Beta-amyloid neurotoxicity in vitro: evidence of oxidative stress but not protection by antioxidants.

Recent data from several groups suggest that the primary mechanism of beta-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert-butyl hydroperoxide) and beta-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not beta-amyloid peptides. Next, we examined whether beta-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or beta-amyloid. In a cell-free system, iron but not beta-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by beta-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to beta-amyloid. The ability of beta-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates beta-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of beta-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to beta-amyloid that underlie its neurodegenerative actions.