Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation

Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kappaB (NF-kappaB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50-200 microM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20-500 microM) or induction of endogenous MT by ZnCl2 (50-200 microM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kappaB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation.     

Identification of an oxidative stress-sensitive protein from Campylobacter jejuni, homologous to rubredoxin oxidoreductase/rubrerythrin

An oxidative stress-sensitive protein was found in the microaerophile Campylobacter jejuni. A novel 27-kDa protein was found to decrease concomitantly with a decrease in viability from either exogenous H(2)O(2) stress or endogenous oxidative stresses in aerobic conditions. Sequence analyses revealed that the 27-kDa protein was identical to Cj0012c in C. jejuni NCTC11168 and its deduced 215 amino acid sequence has similarity to two non-heme iron proteins found in other bacteria, rubredoxin oxidoreductase (Rbo) and rubrerythrin (Rbr). Thus, we designated the protein as Rrc (Rbo/Rbr-like protein of C. jejuni). In H(2)O(2)-treated cells, Western blot analysis showed some bands smaller than Rrc, and RT-PCR showed similar expression of Rrc mRNA to the control without treatment, suggesting that the sensitive response of Rrc to oxidative stress is due to degradation of the protein.     

Diminished Akt phosphorylation in neurons lacking glutathione peroxidase-1 (Gpx1) leads to increased susceptibility to oxidative stress-induced cell death

We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)-/- mice to neuronal apoptosis following mid-cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H2O2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the phospho-inositide 3-kinase [PI3K]-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI3K inhibitor, LY294002. The Gpx1-/- mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1-/- brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia-reperfusion. Our results suggest that the increased susceptibility of Gpx1-/- neurons to H2O2-induced apoptosis and neuronal cell death in vivo following cerebral ischaemia-reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti-apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress-mediated neuropathologies.     

Telomerase activity in response to mild oxidative stress

We have analysed telomerase activity to determine whether it can be modified when BCL-2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl-2 overexpression due to a retroviral infection. Endogenous bcl-2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl-2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl-2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL-2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL-2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL-2 expression when treating patients with ROS (reactive oxygen species) generation by anti-cancer drugs.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.     

Involvement of Hydrogen Peroxide Generated by Polyamine Oxidative Degradation in the Development of Lateral Roots in Soybean

In order to determine whether hydrogen peroxide （H2O2） generated by polyamlne oxidative degradation Is Involved In the development of lateral roots In soybean, the length and the number of lateral roots, the actlvltlea of polyamlne oxldases and dlamlne oxldases, and the endogenous free polyamlne and H2O2 content were analyzed In soybean （Giycine max （Linn.） Merr.） main roots of 2-d-old seedlings after treatments for 2 d with exogenous β-hydroxyethylhydrazine （an Inhibitor of polyamlne oxldases）, H202, putresclne, cyclohexylamlne （an Inhibitor of spermidine synthase） or N,N＇-dimethylthlourea （a scavenger of hydrogen peroxide）.β-hydroxyethylhydrazlne treatment strongly Inhibited the development of lateral roots In soybean seedlings, reduced the activities of polyamine oxldases and dlamlne oxidases, decreased H2O2 levels, and led to the accumulation of endogenous polyamlnes In the main roots. The inhibitory effect of β-hydroxyethylhydrazlne on root development could be alleviated by exogenously applied 10 μmol/L H2O2 （a major product of polyamlne oxidation）. Treatment with cyclohexylamlne and putresclne promoted root growth slightly, but treatment with cyclohexylamlne plus N,N＇dlmethylthlourea or putresclne plus N,N＇-dlmethylthlourea prevented the development of soybean lateral roots. The effects of these treatments on the development of soybean lateral roots were consistent with the changes In endogenous H2O2 levels. These results suggest that the development of soybean lateral roots Is associated with the oxidative degradation of polyamlnes, and that their products, especially H2O2, are likely to play an Important role In the growth of soybean lateral roots.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Redox regulation of endogenous substrate oxidation by cardiac mitochondria

Reactive oxygen species (ROS) play important roles in regulating mitochondrial function, as well as in ischemia-reperfusion injury and cardioprotection. Here we show that, in the absence of exogenous substrates, cardiac mitochondria have a surprisingly large capacity to phosphorylate ADP by oxidizing endogenous substrates, provided that H2O2 is removed from the extramitochondrial environment and a reduced environment is maintained in the matrix. In isolated mitochondria without exogenous substrates, addition of catalase and the membrane-permeant reducing agent N-acetylcysteine (Nac) or the ROS scavenger mercaptopropionyl glycine significantly increased the ability to phosphorylate added ADP, as demonstrated by 1) full recovery of membrane potential (Deltapsi) and matrix volume from ADP-induced dissipation and shrinkage, 2) ADP-dependent increase in O2 consumption, and 3) enhanced rate of ATP synthesis. Removal of extramitochondrial H2O2 by catalase was required to stimulate endogenous substrate oxidation, as shown by the increase in O2 consumption and Deltapsi. This effect was greatly enhanced by addition of Nac or mercaptopropionyl glycine to suppress oxidation-induced ROS increases in the matrix. Theoretical considerations, as well as reversible inhibition of O2 consumption with 3-mercaptopropionic acid and pyruvate in state 3, indicate that these substrates are fatty acids. Under in vivo conditions in which powerful antioxidant conditions are maintained, this mechanism may be important in stimulation of beta-oxidation and ATP production at low levels of extramitochondrial fatty acids. Incapacitation of this mechanism may potentially contribute to mitochondrial dysfunction during oxidative stress.     

Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10?mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel antioxidant proteins that contribute to its increased resistance against oxidative stress.     

Mechanism of adhesion maintenance by methionine sulphoxide reductase in Streptococcus gordonii

Methionine sulphoxide reductase maintains adhesin function during oxidative stress. Using Streptococcus gordonii as a model, we now show the mechanistic basis of adhesin maintenance provided by MsrA. In biofilms, S. gordonii selectively expresses the msrA gene. When the wild-type strain was grown with exogenous hydrogen peroxide (H(2)O(2)), msrA-specific mRNA expression significantly increased, while acid production was unaffected. In the presence of H(2)O(2), a msrA-deletion mutant (ΔMsrA) showed a 6 h delay in lag phase growth, a 30% lower yield of H(2)O(2), significantly greater inhibition by H(2)O(2) on agar plates (reversed by complementation), 30% less adhesion to saliva-coated hydroxyapatite, 87% less biofilm formation and an altered electrophoretic pattern of SspAB protein adhesins. Using mass spectrometry, methionine residues in the Met-rich central region of SspB were shown to be oxidized by H(2)O(2) and reduced by MsrA. In intact wild-type cells, MsrA colocalized with a cell wall-staining dye, and MsrA was detected in both cell wall and cytosolic fractions. To maintain normal adhesion and biofilm function of S. gordonii in response to exogenous oxidants therefore msrA is upregulated, methionine oxidation of adhesins and perhaps other proteins is reversed, and adhesion and biofilm formation is maintained.     

Monoamine oxidase B induces ERK-dependent cell mitogenesis by hydrogen peroxide generation

The mitochondrial enzyme monoamine oxidase (MAO) A and B catalyze the oxidative deamination of various endogenous and exogenous biogenic amines. In the present study, we used human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA to investigate the potential role of hydrogen peroxide (H(2)O(2)) produced by MAO-B isoform as an intracellular messenger involved in regulation of cell signaling and function. The MAO substrate tyramine induced tyrosine phosphorylation of Shc, ERK activation, and an increase in DNA synthesis in HEK 293 expressing MAO-B, but not in wild type HEK 293 cells, which do not express MAO. Tyramine effects were fully prevented by cell pretreatment with the MAO inhibitor pargyline or the antioxidant N-acetylcysteine. These results show that MAO-B induces MAPK/ERK activation and cell mitogenesis through H(2)O(2) production.     

Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts

Perturbation of oxidant/antioxidant cellular balance, induced by cellular metabolism and by exogenous sources, causes deleterious effects to proteins, lipids, and nucleic acids, leading to a condition named "oxidative stress" that is involved in several diseases, such as cancer, ischemia-reperfusion injury, and neurodegenerative disorders. Among the exogenous agents, both H(2)O(2) and hyperthermia have been implicated in oxidative stress promotion linked with the activation of apoptotic or necrotic mechanisms of cell death. The goal of this work was to better understand the involvement of some stress-related proteins in adaptive responses mounted by human fibroblasts versus the oxidative stress differently induced by 42 degrees C hyperthermia or H(2)O(2.) The research was developed, switching off inducible nitric oxide synthase (iNOS) expression through antisense oligonucleotide transfection by studying the possible coregulation in the expression of HSP32 (also named HO-1), HSP70, and iNOS and their involvement in the induction of DNA damage. Several biochemical parameters, such as cell viability (MTT assay), cell membrane integrity (lactate dehydrogenase release), reactive oxygen species formation, glutathione levels, immunocytochemistry analysis of iNOS, HSP70, and HO-1 levels, genomic DNA fragmentation (HALO/COMET assay), and transmembrane mitochondrial potential (deltaPsi) were examined. Cells were collected immediately at the end of the stress-inducing treatment. The results, confirming the pleiotropic function of i-NOS, indicate that: (i). HO-1/HSP32, HSP70, and iNOS are finely tuned in their expression to contribute all together, in human fibroblasts, in ameliorating the resistance to oxidative stress damage; (ii). ROS exposure, at least in hyperthermia, in human fibroblasts contributes to growth arrest more than to apoptosis activation; and (iii). mitochondrial dysfunction, in presence of iNOS inhibition seems to be clearly involved in apoptotic cell death of human fibroblasts after H(2)O(2) treatment, but not after hyperthermia.     

Mitochondrial DNA damage is sensitive to exogenous H(2)O(2) but independent of cellular ROS production in prostate cancer cells

Intrinsic oxidative stress through enhanced production of reactive oxygen species (ROS) in prostate and other cancers may contribute to cancer progression due to its stimulating effect on cancer growth. In this study, we investigate differential responses to exogenous oxidative stimuli between aggressive prostate cancer and normal cell lines and explore potential mechanisms through interactions between cytotoxicity, cellular ROS production and oxidative DNA damage. The circular, multi-copy mitochondrial DNA (mtDNA) is used as a sensitive surrogate to oxidative DNA damage. We demonstrate that exogenous H(2)O(2) induces preferential cytotoxicity in aggressive prostate cancer than normal cells; a cascade production of cellular ROS, composed mainly of superoxide (O(2)(-)), is shown to be a critical determinant of H(2)O(2)-induced selective toxicity in cancer cells. In contrast, mtDNA damage and copy number depletion, as measured by a novel two-phase strategy of the supercoiling-sensitive qPCR method, are very sensitive to exogenous H(2)O(2) exposure in both cancer and normal cell lines. Moreover, we demonstrate for the first time that the sensitive mtDNA damage response to exogenous H(2)O(2) is independent of secondary cellular ROS production triggered by several ROS modulators regardless of cell phenotypes. These new findings suggest different mechanisms underpinning cytotoxicity and DNA damage induced by oxidative stress and a susceptible phenotype to oxidative injury associated with aggressive prostate cancer cells in vitro.     

Oxidation of Imported or Endogenous Carbohydrates by Isolated Chloroplasts from Green Pepper Fruits

Recently, we demonstrated that intact chloroplasts isolated from green pepper (Capsicum annum L.) fruits use exogenous glucose-6-phosphate (Glc-6-P) as the most efficient precursor for starch biosynthesis (O. Batz, R. Scheibe, H.E. Neuhaus [1995] Planta 196: 50-57). Here we demonstrate that these chloroplasts transport this hexose phosphate in counter-exchange for orthophosphate. By measuring the release of 14CO2 from [1-14C]Glc-6-P, we show that isolated fruit chloroplasts also use exogenous Glc-6-P as a substrate for the oxidative pentose-phosphate pathway. The rate of decarboxylation appears to be linear with time and is significantly reduced in the presence of Triton X-100, indicating that the reaction is dependent on plastid integrity. Pyruvate has been identified as a positive effector for flux through the oxidative pentose-phosphate pathway. However, the highest rates of Glc-6-P-driven oxidative pentosephosphate pathway activity are achieved in the presence of nitrite, 2-oxoglutarate, and glutamine, indicating a strong interaction between nitrogen metabolism and this pathway. In addition, we show that carbohydrates liberated during starch mobilization are used as substrates for the oxidative pentose-phosphate pathway. Orthophosphate was found to act as an activator for the observed 14CO2 release from carbohydrates formerly bound as starch. In this context, we demonstrate that exogenous Glc-6-P competes with endogenous carbohydrates. A possible interaction between exogenous and endogenous carbohydrates is discussed with respect to altered levels of carbohydrates during fruit development.     

Recovery of porcine aortic endothelial cell prostaglandin synthesis following inhibition by sublethal concentrations of hydrogen peroxide

Confluent monolayers of porcine aortic endothelial cells exposed for 10 min to 100 microM H2O2 lose their capacity to produce prostaglandins in response to addition of saturating exogenous arachidonic acid. Significant recovery of prostaglandin I2 and E2 synthesis occurred within 3 h and full enzymatic capacity returned by 6 h. Reducing the injury by exposure to half the amount of H2O2 allowed prostaglandin I2 production to recover to a greater extent in 3 h, while cells exposed for 60 min to either 0.5 or 1.0 mM H2O2 demonstrated no recovery. Pre-treatment with either actinomycin D or cycloheximide also prevented recovery following exposure to 100 microM peroxide. Injured cells did not recover when incubated with balanced salts after removal of peroxide, while incubation with medium 199 allowed for the complete return of synthetic capacity. Addition of 1% fetal calf serum in medium 199 did not facilitate recovery. Production of prostaglandins from endogenous arachidonic acid, released by either bradykinin or the ionophore A23187, was also inhibited by H2O2 exposure, however, full recovery of this stimulated synthesis occurred within 3 h. Cycloheximide pre-treatment completely inhibited recovery of bradykinin-induced prostaglandin I2 synthesis. These data demonstrate that sublethal concentrations of H2O2 irreversibly inactivate fatty acid cyclooxygenase and that synthesis of new enzyme is required for recovery. This return of activity occurs more rapidly for production of prostaglandins from endogenous arachidonic acid compared with production following addition of exogenous substrate.     

Oxidative stress in hypercholesterolemic LDL (low-density lipoprotein) receptor knockout mice is associated with low content of mitochondrial NADP-linked substrates and is partially reversed by citrate replacement

We have previously proposed that hypercholesterolemic LDL receptor knockout (k/o) mice mitochondria possess a lower antioxidant capacity due to a large consumption of reducing equivalents from NADPH to sustain high rates of lipogenesis. In this work, we tested the hypothesis that this k/o mice mitochondrial oxidative stress results from the depletion of NADPH-linked substrates. In addition, the oxidative stress was further characterized by showing a lower mitochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to controls. The activity of the antioxidant enzyme system glutathione reductase/peroxidase did not differ in k/o and control mitochondria. The faster spontaneous oxidation of endogenous NADPH in the k/o mitochondria was prevented by the addition of exogenous catalase, indicating that this oxidation is mediated by mitochondrially generated H(2)O(2). The higher rate of H(2)O(2) production was also prevented by the addition of exogenous isocitrate that maintains NADP fully reduced. The hypothesis that high rates of lipogenesis in the k/o cells decrease mitochondrial NADPH/NADP(+) ratio due to consumption of NADPH-linked substrates was supported by two findings: (i) oxygen consumption supported by endogenous NAD(P)H-linked substrates was slower in k/o than in control mitochondria, but was similar in the presence of exogenous isocitrate; (ii) in vivo treatment of k/o mice with sodium citrate/citric acid drinking solution for 2 weeks partially restored both the rate of oxygen consumption supported by NAD(P)H-linked substrates and the mitochondrial capacity to sustain reduced NADPH. In conclusion, the data demonstrate that the mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate.