Pro- or anti-oxidant manganese: a suggested mechanism for reconciliation

The neurodegeneration induced by manganese has been attributed to its ability to undergo redox cycling, and catalysis of reactive oxygen species (ROS) formation, as with other transition metals. However, the characterization of manganese as a pro-oxidant is confounded by increasing evidence that the metal may scavenge superoxide anions and protect cells from oxidative damage. The current study was designed to address conflicting reports pertaining to the oxidative capacity of manganese. We found that the metal has distinctive redox dynamics in which the divalent reduced form, unlike iron, possessed no intrinsic oxidative capacity. The apparent ability of Mn(2+) to promote the formation of ROS within a cortical mitochondrial-synaptosomal fraction was quenched by the depletion of contaminating nanomolar concentrations of trivalent metals. The addition of manganic ions at trace concentrations dose-dependently restored the oxidative capacity attributed to divalent manganese, whereas the presence of the ferric ion retarded the rate of ROS generation. This result was paralleled by the spectrophotometric demonstration that the kinetics of iron oxidation is accelerated by trivalent but not divalent manganese. The markedly different capacities of the lower and higher valence states of manganese to promote free-radical formation in cortical fractions and to modulate the process of iron oxidation may account for earlier contradictory reports of anti- and pro-oxidant properties of manganese.     

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.     

Induction of oxidative stress by humic acid through increasing intracellular iron: a possible mechanism leading to atherothrombotic vascular disorder in blackfoot disease

Humic acid (HA), a potential toxin that has penetrated the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as an etiological factor of this disease. In this study, we investigated the effects of HA on the generation of reactive oxygen species (ROS) in cultured human umbilical vein endothelial cells (HUVECs). The generation of ROS was monitored by flow cytometry. Pretreatment of HUVECs with HA induced reactive oxygen species in a dose- and time-dependent manner. Xanthine oxidase inhibitor (Allopurinol), NADPH oxidase inhibitor (diphenylene iodomium) and calcium chelator (BAPTA) could not reduce the generation of ROS. Protein kinase C inhibitor (H7) could reduce the generation of ROS slightly, but the intracellular antioxidant glutathione monoethyl ester and the iron chelator desferrioxamine (DFO) could inhibit the generation of ROS completely. HA also enhanced the expression of ferritin and induced intracellular chelatable iron; however, HA reduced the expression of transferrin receptor. Pretreatment with DFO inhibited HA-mediated increases of ferritin synthesis and intracellular chelatable iron, but caused recovery of the inhibitory effect on transferrin receptor. Cotreatment with iron and HA induced more ROS and intracellular chelatable iron than iron or HA treatment alone. Furthermore, HA enhanced the accumulation of iron in endothelial cells. These data demonstrate that HA can increase the generation of ROS through enhancing the accumulation of intracellular iron. Taken together, our findings suggest that iron mediates HA-associated oxidative stress in endothelial cells, which may be a possible mechanism leading to atherothrombotic vascular injury observed for patients with blackfoot disease.     

Studies on Auxin Protectors. IV. The Effect of Manganese on Auxin Protector-I of the Japanese Morning Glory

Auxin protector-I of the Japanese morning glory is inactivated by manganese. Experiments carried out in vitro indicate that in the absence of oxygen the manganic, but not the manganous, ion rapidly inactivates the protector. It is clear from these, and other data described in this report, and the results of other workers, that in the presence of oxygen, manganese accelerates auxin inactivation by means of 2 separate and distinct mechanisms: 1) manganese catalyzes the oxidation of auxin protectors, and 2) following the inactivation of the protectors, or in the absence of protectors, accelerates the oxidation of indoleacetic acid by endogenous peroxidases.     

Quercetin protects C6 glial cells from oxidative stress induced by tertiary-butylhydroperoxide

The anti-oxidant and cyto-protective activity of quercetin against tertiary-butylhydroperoxide (t-BOOH) induced oxidative stress on C6 glial cells is reported. Exposure of the cells to t-BOOH resulted in a significant increase in cytotoxicity, reactive oxygen species (ROS) generation and lipid peroxidation. There was a significant increase in DNA strand breaks and fall in reduced GSH levels in cells exposed to t-BOOH. A significant increase in calcium ion influx was noticed in cells exposed to t-BOOH. Pre-treatment of cells with quercetin, vitamin C (vit C), Trolox, and deferoxamine (DFO) significantly inhibited t-BOOH induced cytotoxicity and ROS generation. Pretreatment of cells with quercetin, Trolox and DFO inhibited the DNA damage, maintained higher GSH levels and prevented calcium influx significantly. Although vit C protected the cells from cytotoxicity induced by t-BOOH, the intracellular Ca²⁺ levels were significantly higher than the control cells. However, anti-oxidants like butylated hydroxy toluene (BHT), vitamin E (vit E), N-acetyl cysteine (NAC) did not have significant cytoprotection against t-BOOH induced oxidative injury in C6 glial cells.     

Towards the prevention of potential aluminum toxic effects and an effective treatment for Alzheimer's disease

In 1991, treatment with low dose intramuscular desferrioxamine (DFO), a trivalent chelator that can remove excessive iron and/or aluminum from the body, was reported to slow the progression of Alzheimer's disease (AD) by a factor of two. Twenty years later this promising trial has not been followed up and why this treatment worked still is not clear. In this critical interdisciplinary review, we provide an overview of the complexities of AD and involvement of metal ions, and revisit the neglected DFO trial. We discuss research done by us and others that is helping to explain involvement of metal ion catalyzed production of reactive oxygen species in the pathogenesis of AD, and emerging strategies for inhibition of metal-ion toxicity. Highlighted are insights to be considered in the quests to prevent potentially toxic effects of aluminum toxicity and prevention and intervention in AD.     

Carbon monoxide and bilirubin from heme oxygenase-1 suppresses reactive oxygen species generation and plasminogen activator inhibitor-1 induction

Heme oxygenase-1 (HO-1) responds to a variety of oxidative stresses. We examined whether HO-1 expression influences pro-thrombotic processes, in which the involvement of oxidative stress has been reported. Since HO-1 knockout mice with a C57/BL6J background were not viable, embryonic cells from HO-1 deficient mice (E11.5) were used. Cell viability, the level of plasminogen activator inhibitor-1 (PAI-1) expression and reactive oxygen species (ROS) generation of HO-1 deficient cells in response to the exposures to hydrogen peroxide and oxidized LDL were compared to those with wild-type cells. We also examined the effects of glutathione (GSH), desferrioxamine (DFO) and diphenyleneiodonium (DPI: an NADPH oxidase inhibitor) as well as of the HO reaction products, bilirubin (BR) and carbon monoxide (CO) on PAI-1 expression and ROS generation. PAI-1 expression and ROS generation were markedly elevated in HO-1 deficient cells compared to wild-type cells. Exposure to oxidized LDL significantly elevated PAI-1 expression and ROS production in HO-1 deficient cells. Interestingly, these increases in HO-1 deficient cells were significantly lowered by BR, CO, GSH and DPI while DFO had little effect. Furthermore, BR and CO were effective to improve viabilities of HO-1 deficient cells. These results suggest that HO-1 may be required to suppress ROS generation and the production of pro-thrombotic molecules such as PAI-1.     

Degradation of lyophilized lipid/DNA complexes during storage: The role of lipid and reactive oxygen species

The presence of trace amounts of metal ions in nonviral vector formulations can significantly affect the stability of lipid/DNA complexes (lipoplexes) during acute freeze-drying. The goal of the present study was to evaluate the generation of reactive oxygen species (ROS) in dried formulations of lipoplexes and in their individual components (lipid or naked DNA). The experiments were conducted in the presence or absence of a transition metal (Fe²⁺). Lipoplexes and their individual components were formulated in trehalose and subjected to lyophilization and stored for a period of up to 2 months at + 60 °C. Physico-chemical characteristics and biological activity were evaluated at different time intervals. Generation of ROS during storage was determined by adding a fluorescence probe to the formulations prior to freeze-drying. We also monitored the formation of thiobarbituric reactive substances (TBARS). Our results show that ROS and TBARS form during storage in the dried state. Our findings also suggest that degradation is more rapid in the presence of lipid, even in the absence of metal. We also showed that dried naked DNA formulations are more stable without the lipid component. Effective strategies are then needed to minimize the formation and accumulation of oxidative damage of lipoplexes during storage.     

Drug resistance to 5-FU linked to reactive oxygen species modulator 1

While acute oxidative stress triggers cell apoptosis or necrosis, persistent oxidative stress induces genomic instability and has been implicated in tumor progression and drug resistance. In a previous report, we demonstrated that reactive oxygen species modulator 1 (Romo1) expression was up-regulated in most cancer cell lines and suggested that increased Romo1 expression might confer chronic oxidative stress to tumor cells. In this study, we show that enforced Romo1 expression induces reactive oxygen species (ROS) production in the mitochondria leading to massive cell death. However, tumor cells that adapt to oxidative stress by increasing manganese superoxide dismutase (MnSOD), Prx I, and Bcl-2 showed drug resistance to 5-FU. To elucidate the relationship between 5-FU-induced ROS production and Romo1 expression, Romo1 siRNA was used to inhibit 5-FU-triggered Romo1 induction. Romo1 siRNA treatment efficiently blocked 5-FU-induced ROS generation, demonstrating that 5-FU treatment stimulated ROS production through Romo1 induction. Based on these results we suggest that cellular adaptive response to Romo1-induced ROS is another mechanism of drug resistance to 5-FU and Romo1 expression may provide a new clinical implication in drug resistance of cancer chemotherapy.     

Formation of reactive oxygen species in rat epithelial cells upon stimulation with fly ash

Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 microm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (0.1 microg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.     

Relationship between trace metal concentration and antioxidative activity of ancient rice bran (red and black rice) and a present-day rice bran (Koshihikari).

Antioxidative activity and polyphenol and trace metal content in bran from ancient rice varieties (red and black rice) and a present-day variety of rice (Koshihikari) were measured. The antioxidative properties of rice bran in terms of scavenging and quenching activity for reactive oxygen species (ROS), including superoxide anion radicals (*O(2)(-)), hydroxyl radicals (*OH), singlet oxygen ((1)O(2)) and lipid peroxide (LOO*), correlated well with polyphenol and trace metal content. In particular, the possibly that Mn content greatly contributes to the antioxidative properties of rice bran was revealed.     

Effect of Anions on the Binding and Oxidation of Divalent Manganese and Iron in Modified Bacterial Reaction Centers

The influence of different anions on the binding and oxidation of manganous and ferrous cations was studied in four mutants of bacterial reaction centers that can bind and oxidize these metal ions. Light-minus-dark difference optical and electron paramagnetic resonance spectroscopies were applied to monitor electron transfer from bound divalent metal ions to the photo-oxidized bacteriochlorophyll dimer in the presence of five different anions. At pH 7, bicarbonate was found to be the most effective for both manganese and iron binding, with dissociation constants around 1 μM in three of the mutants. The pH dependence of the dissociation constants for manganese revealed that only bicarbonate and acetate were able to facilitate the binding and oxidation of the metal ion between pH 6 and 8 where the tight binding in their absence could not otherwise be established. The data are consistent with two molecules of bicarbonate or one molecule of acetate binding to the metal binding site. For ferrous ion, the binding and oxidation was facilitated not only by bicarbonate and acetate, but also by citrate. Electron paramagnetic resonance spectra suggest differences in the arrangement of the iron ligands in the presence of the various anions.     

Reactive oxygen species generation and signaling in plants

The introduction of molecular oxygen into the atmosphere was accompanied by the generation of reactive oxygen species (ROS) as side products of many biochemical reactions. ROS are permanently generated in plastids, peroxisomes, mitochiondria, the cytosol and the apoplast. Imbalance between ROS generation and safe detoxification generates oxidative stress and the accumulating ROS are harmful for the plants. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses. Here, we summarize the generation of ROS in the different cellular compartments and the signaling processes which are induced by ROS.Keyword: reactive oxygen species, signal transduction, plastids     

Desferoxamine and ethyl-3,4-dihydroxybenzoate protect myocardium by activating NOS and generating mitochondrial ROS

Protection from a prolyl hydroxylase domain-containing enzyme (PHD) inhibitor, desferoxamine (DFO), was recently reported to be dependent on production of reactive oxygen species (ROS). Ischemic preconditioning triggers the protected state by stimulating nitric oxide (NO) production to open mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels, generating ROS required for protection. We tested whether DFO and a second PHD inhibitor, ethyl-3,4-dihydroxybenzoate (EDHB), might have similar mechanisms. EDHB and DFO increased ROS generation by 50-75% (P < 0.001) in isolated rabbit cardiomyocytes. This increase after EDHB exposure was blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), an NO synthase (NOS) inhibitor; ODQ, a guanylyl cyclase antagonist; and Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate Rp isomer, a PKG blocker, thus implicating the NO pathway in EDHB's signaling. Glibenclamide, a nonselective K(ATP) channel blocker, or 5-hydroxydecanoate, a selective mitoK(ATP) channel antagonist, also prevented EDHB's ROS production, as did blockade of mitochondrial electron transport with myxothiazol. NOS is activated by Akt. However, neither wortmannin, an inhibitor of phosphatidylinositol-3-kinase, nor Akt inhibitor blocked EDHB-induced ROS generation, indicating that EDHB initiates signaling downstream of Akt. DFO also increased ROS production, and this effect was blocked by ODQ, 5-hydroxydecanoate, and N-(2-mercaptopropionyl)glycine, an ROS scavenger. DFO increased cardiomyocyte production of nitrite, a metabolite of NO, and this effect was blocked by an inhibitor of NOS. DFO also spared ischemic myocardium in intact hearts. This infarct-sparing effect was blocked by ODQ, L-NAME, and N-(2-mercaptopropionyl)glycine. Hence, DFO and EDHB stimulate NO-dependent activation of PKG to open mitoK(ATP) channels and produce ROS, which act as second messengers to trigger entrance into the preconditioned state.     

Screening of dietary antioxidants against mitochondria-mediated oxidative stress by visualization of intracellular redox state

Mitochondrial impairment and the resulting generation of reactive oxygen species (ROS) have been associated with aging and its related pathological conditions. Recently, dietary antioxidants have gained significant attention as potential preventive and therapeutic agents against ROS-generated aging and pathological conditions. We previously demonstrated that food-derived antioxidants prevented intracellular oxidative stress under proteasome inhibition conditions, which was attributed to mitochondrial dysfunction and ROS generation, followed by cell death. Here, we further screened dietary antioxidants for their activity as redox modulators by visualization of the redox state using Redoxfluor, a fluorescent protein redox probe. Direct alleviation of ROS by antioxidants, but not induction of antioxidative enzymes, prevented mitochondria-mediated intracellular oxidation. The effective antioxidants scavenged mitochondrial ROS and suppressed cell death. Our study indicates that redox visualization under mitochondria-mediated oxidative stress is useful for screening potential antioxidants to counteract mitochondrial dysfunction, which has been implicated in aging and the pathogenesis of aging-related diseases.     

Oxidative stress and antioxidant defenses in ethanol-induced cell injury

Although in the past several mechanisms and factors have been proposed to be responsible for alcoholic liver disease (ALD), at present the involvement of oxygen free radicals and consequently of oxidative stress has acquired remarkable credit. In numerous experimental studies it has been shown the occurrence of alcohol-induced generation of oxygen- and ethanol-derived free radicals through different pathways and from different sources. Mitochondria appear to be both an important source of reactive oxygen species (ROS) and also a primary target of ethanol-induced damage. The consistent induction of the mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) observed in experimental animals after acute and chronic ethanol administration has all the characteristics of a "stress response" to an oxidative insult.     

Aluminum induced oxidative events and its relation to inflammation: a role for the metal in Alzheimer's disease.

Aluminum (Al) is a simple trivalent cation incapable of redox changes. The toxicity of the metal has been the subject of much controversy in the past few decades. Although it has been generally believed that the metal is innocuous to human health, a causal role for Al has been established in dialysis dementia (Alfrey et al., 1976), osteomalacia (Bushinsky et al., 1995) and microcytic anemia without iron deficiency (Touam et al., 1983). Aluminum has also been implicated in Alzheimer's disease (AD) although a direct causal role has not been determined. The exact mechanism of Al toxicity is not known. However, there are several lines of evidence that show the metal's capacity to exacerbate oxidative events. The present review is intended to propose a coherent pathway linking Al-induced oxidative events to Alzheimer's disease. The preliminary segment is an introduction to reactive oxygen species and their potential involvement in the pathogenesis of AD and the generation of an inflammatory response. Evidence on the relation between AD and inflammatory processes is also presented. The epidemiological and clinical evidence of Al neurotoxicity is summarized in the second section of the review. Finally, a hypothesis indicating that aluminum can exacerbate AD by activating ROS generation and initiation of an inflammatory cascade is presented.     

Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions. Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls. Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances. The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride. Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM. The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol. When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis. To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen. The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1). In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.     

Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung

Exposure of newborn mice to high inspired oxygen elicits a distinct phenotype of compromised alveolar and vascular development, although lethality during long-term exposure is lower in newborns compared to adults. As the effects of hyperoxia are mediated by excessive reactive oxygen species (ROS) generation, we hypothesized that newborn mice may exhibit enhanced expression of antioxidant defenses or attenuated ROS generation compared with adults. We measured subcellular oxidant responses to acute hyperoxia in lung slices and alveolar epithelial cells at varying time points during postnatal murine lung development. Oxidant stress was assessed using RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. In contrast to newborn resistance to oxygen-induced mortality, cells of lung slices from younger mice demonstrated exaggerated mitochondrial matrix oxidant stress compared to adults, whereas oxidant stress responses in the cytosol were absent. Cell death in lung slices from newborn mice exposed to 48 h of hyperoxia was also greater than for adults. Consistent with these findings, expression of antioxidant enzymes in newborn lungs was lower than in adults, and induction of antioxidant levels and activity during 24 h of in vivo exposure was absent. However, expression of the reactive oxygen species-generating enzyme NADPH oxidase 1 was increased with hyperoxic exposure in the young but not the adult lung. Collectively, these results suggest that the greater lethality in adult animals may be more likely attributed to processes such as inflammation than to differences in antioxidant defenses. Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses.     

Conformational studies of A23187 with mono-, di- and trivalent metal ions by circular dichroism spectroscopy

CD studies carried out on A23187 indicate a solvent-dependent conformation for the free acid. Alkali metal ions were found to bind to the ionophore weakly. Divalent metal ions such as Mg2+, Ca2+, Sr2+, Ba2+ and Co2+ and trivalent lanthanide metal ions like La3+ were found to form predominantly 2:1 (ionophore-metal ion) complexes at low concentrations of metal ions, but both 2:1 and 1:1 complexes were formed with increasing salt concentration. Mg2+ and Co2+ exhibit similar CD behaviour that differs from that observed for the other divalent and lanthanide metal ions. The structure of 2:1 complexes involves two ligand molecules coordinated to the metal ion through the carboxylate oxygen, benzoxazole nitrogen and keto-pyrrole oxygen from each ligand molecule along with one or more solvent molecules. Values of the binding constant were determined for 2:1 complexes of the ionophore with divalent and lanthanide metal ions.