| 1. |
- Aldridge, Ruth E., et al.
(författare)
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Eosinophil peroxidase produces hypobromous acid in the airways of stable asthmatics
- 2002
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 33:6, s. 847-856
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Tidskriftsartikel (refereegranskat)abstract
- Eosinophil peroxidase and myeloperoxidase use hydrogen peroxide to produce hypobromous acid and hypochlorous acid. These powerful oxidants may damage the lungs if they are produced as part of the inflammatory response in asthma. The aim of this study was to determine if peroxidases generate hypohalous acids in the airways of individuals with stable asthma, and if they affect lung function. Sputum was induced from patients with mild to moderate asthma and from healthy controls. Eosinophil peroxidase, myeloperoxidase, chlorinated and brominated tyrosyl residues, and protein carbonyls were measured in sputum supernatants. Eosinophil peroxidase protein was significantly elevated in asthmatic subjects whereas myeloperoxidase protein was not. There was significantly more 3-bromotyrosine (Br-Tyr) in proteins from the sputum of asthmatics compared to controls (0.79 vs. 0.23 mmol Br-Tyr/mol Tyr; medians p < .0001). Levels of 3-chlorotyrosine (0.23 vs. 0.14 mmol Cl-Tyr/mol Tyr; medians p = .11) and protein carbonyls (0.347 vs. 0.339 nmol/mg protein; medians p = .56) were not significantly increased in asthmatics. Levels of 3-bromotyrosine were strongly correlated with eosinophil peroxidase protein (r = 0.79, p < .0001). There were no significant correlations between the markers of oxidative stress and lung function. We conclude that eosinophil peroxidase produces substantial amounts of hypobromous acid in the airways of stable asthmatics. Although this highly reactive oxidant is a strong candidate for exacerbating inflammatory tissue damage in the lung, its role in asthma remains uncertain.
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| 2. |
- Arnér, Elias S. J., et al.
(författare)
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Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex
- 2001
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Ingår i: Free Radical Biology & Medicine. - : Elsevier. - 0891-5849 .- 1873-4596. ; 31:10, s. 1170-1178
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Tidskriftsartikel (refereegranskat)abstract
- Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.
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| 3. |
- Brunk, Ulf, 1937-, et al.
(författare)
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Lipofuscin : Mechanisms of age-related accumulation and influence on cell function
- 2002
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 33:5, s. 611-619
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Tidskriftsartikel (refereegranskat)abstract
- The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occuring with a rate inversely related to longevity. Lipofuscin is an intralysosomal, polymeric substance, primarily composed of cross-linked protein residues, formed due to iron-catalyzed oxidative processes. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic cells is inevitable, whereas proliferative cells efficiently dilute it during division. The rate of lipofuscin formation can be experimentally manipulated. In cell culture models, oxidative stress (e.g., exposure to 40% ambient oxygen or low molecular weight iron) promotes lipofuscin accumulation, whereas growth at 8% oxygen and treatment with antioxidants or iron-chelators diminish it. Lipofuscin is a fluorochrome and may sensitize lysosomes to visible light, a process potentially important for the pathogenesis of age-related macular degeneration. Lipofuscin-associated iron sensitizes lysosomes to oxidative stress, jeopardizing lysosomal stability and causing apoptosis due to release of lysosomal contents. Lipofuscin accumulation may also diminish autophagocytotic capacity by acting as a sink for newly produced lysosomal enzymes and, therefore, interfere with recycling of cellular components. Lipofuscin, thus, may be much more directly related to cellular degeneration at old age than was hitherto believed.
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| 4. |
- Daré, Elisabetta, et al.
(författare)
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Methylmercury and H2O2 provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis
- 2001
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Ingår i: Free Radical Biology & Medicine. - Linköping : Elsevier. - 0891-5849 .- 1873-4596. ; 30:12, s. 1347-1356
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Tidskriftsartikel (refereegranskat)abstract
- Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H2O2) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H2O2 preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H2O2 stimulated divergent pathways, with caspases being activated only by H2O2. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H2O2, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H2O2. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.
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| 5. |
- Doulias, Paschalis-Thomas, et al.
(författare)
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Endosomal and lysosomal effects of desferrioxamine : Protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest
- 2003
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 35:7, s. 719-728
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Tidskriftsartikel (refereegranskat)abstract
- The role of endosomal/lysosomal redox-active iron in H2O 2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 ╡M) inhibited cell proliferation in a fluid-phase endocytosis- dependent manner. Flow cytometric analysis of cells exposed to 100 ╡M DFO for 24 h showed that the cell cycle was transiently interrupted at the G 2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O 2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible.
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| 6. |
- Hellsten, Y, et al.
(författare)
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Allantoin formation and urate and glutathione exchange in human muscle during submaximal exercise.
- 2001
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 31:11, s. 1313-22
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Tidskriftsartikel (refereegranskat)abstract
- Seven males performed two exhaustive cycling bouts (EX1 and EX2) at a work-rate of 90% of maximal oxygen uptake, separated by 60 min. During EX1 there was a significant accumulation of urate (from 0.16 +/- 0.02 to 0.27 +/- 0.03 micromol/kg d.w.) and allantoin (from 0.39 +/- 0.05 to 0.69 +/- 0.14 micromol/kg d.w.) in the muscle. An uptake of urate was observed in early recovery from EX1 (0-9 min: 486 +/- 136 micromol; p <.05). There was no exchange of total glutathione or cysteine over the muscle either during or after exercise, and muscle and plasma total glutathione remained unaltered (p <.05). The glycogen levels were lowered by 40% at the onset of EX2, yet the level of oxidative stress in EX1 and EX2 was similar as evidenced by a similar increase in muscle allantoin in both exercise bouts. The data suggest that urate is utilized as antioxidant in human skeletal muscle and that reactive oxygen species are formed in muscle during intense submaximal exercise. No net exchange of glutathione appears to occur over the muscle either at rest, during exercise or in recovery. Moreover, when an exhaustive exercise bout is repeated with lowered glycogen levels, the level of oxidative stress is not different than that of the first bout.
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| 7. |
- Löntz, Werner, et al.
(författare)
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Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha
- 1995
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 18:2, s. 349-355
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Tidskriftsartikel (refereegranskat)abstract
- Because reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of the antioxidant enzyme superoxide dismutase was studied in psoriatic skin tissue. By using reverse transcription-PCR we found similar expression of copper, zinc superoxide dismutase (CuZnSOD) in the involved vs. uninvolved psoriatic skin. In contrast, the level of the manganese superoxide dismutase (MnSOD) mRNA message was consistently higher in lesional psoriatic skin as compared to adjacent uninvolved skin and healthy control skin. Parallel investigation of those cytokines that are thought to be direct or indirect inducers of the MnSOD activity revealed an increased mRNA expression of IL-1 beta, TNF-alpha, and GM-CSF in lesional psoriatic skin. To study if these cytokines exert a direct effect on dismutase expression in epidermal cells, human keratinocytes in culture were challenged with IL-1 beta, TNF-alpha, and GM-CSF. It was found that IL-1 beta and TNF-alpha, but not GM-CSF, induced the mRNA expression of MnSOD, and an additive effect was demonstrated for the two former cytokines. Further, the expression of both CuZnSOD and MnSOD transcripts was similar in cultured keratinocytes maintained at low differentiation (low Ca2+ medium) and cells forced to terminal differentiation (by high Ca2+ medium). Our results indicate that the abnormal expression of MnSOD mRNA in lesional psoriatic skin is not directly linked to the pathologic state of keratinocyte differentiation in the skin. It seems more likely that the cutaneous overexpression of MnSOD in psoriatic epidermis represents a protective cellular response evoked by cytokines released from inflammatory cells invading the diseased skin.
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| 8. |
- Mudway, I S, et al.
(författare)
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Differences in basal airway antioxidant concentrations are not predictive of individual responsiveness to ozone : a comparison of healthy and mild asthmatic subjects
- 2001
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Ingår i: Free Radical Biology & Medicine. - : Elsevier. - 0891-5849 .- 1873-4596. ; 31:8, s. 962-974
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Tidskriftsartikel (refereegranskat)abstract
- The air pollutant ozone induces both airway inflammation and restrictions in lung function. These responses have been proposed to arise as a consequence of the oxidizing nature of ozone, depleting endogenous antioxidant defenses with ensuing tissue injury. In this study we examined the impact of an environmentally relevant ozone challenge on the antioxidant defenses present at the surface of the lung in two groups known to have profound differences in their antioxidant defense network: healthy control (HC) and mild asthmatic (MA) subjects. We hypothesized that baseline differences in antioxidant concentrations within the respiratory tract lining fluid (RTLF), as well as induced responses, would predict the magnitude of individual responsiveness. We observed a significant loss of ascorbate (ASC) from proximal (-45.1%, p <.01) and distal RTLFs (-11.7%, p <.05) in healthy subjects 6 h after the end of the ozone challenge. This was associated (Rs, -0.71, p <.01) with increased glutathione disulphide (GSSG) in these compartments (p =.01 and p <.05). Corresponding responses were not seen in asthmatics, where basal ASC concentrations were significantly lower (p <.01) and associated with elevated concentrations of GSSG (p <.05). In neither group was any evidence of lipid oxidation seen following ozone. Despite differences in antioxidant levels and response, the magnitude of ozone-induced neutrophilia (+20.6%, p <.01 [HC] vs. +15.2%, p =.01 [MA]) and decrements in FEV(1) (-8.0%, p <.01 [HC] vs. -3.2%, p <.05 [MA]) did not differ between the two groups. These data demonstrate significant differences between the interaction of ozone with RTLF antioxidants in MA and HC subjects. These responses and variations in basal antioxidant defense were not, however, useful predictive markers of group or individual responsiveness to ozone.
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| 9. |
- Persson, Lennart, 1961-, et al.
(författare)
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Prevention of oxidant-induced cell death by lysosomotropic iron chelators
- 2003
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 34:10, s. 1295-1305
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Tidskriftsartikel (refereegranskat)abstract
- Intralysosomal iron powerfully synergizes oxidant-induced cellular damage. The iron chelator, desferrioxamine (DFO), protects cultured cells against oxidant challenge but pharmacologically effective concentrations of this drug cannot readily be achieved in vivo. DFO localizes almost exclusively within the lysosomes following endocytic uptake, suggesting that truly lysosomotropic chelators might be even more effective. We hypothesized that an amine derivative of α-lipoamide (LM), 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (α-lipoic acid-plus [LAP]; pKa = 8.0), would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of this agent would interact with intralysosomal iron, preventing oxidant-mediated cell damage. Using a thiol-reactive fluorochrome, we find that reduced LAP does accumulate within the lysosomes of cultured J774 cells. Furthermore, LAP is approximately 1,000 and 5,000 times more effective than LM and DFO, respectively, in protecting lysosomes against oxidant-induced rupture and in preventing ensuing apoptotic cell death. Suppression of lysosomal accumulation of LAP (by ammonium-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic resonance reveals that the intracellular generation of hydroxyl radical following addition of hydrogen peroxide to J774 cells is totally eliminated by pretreatment with either DFO (1 mM) or LAP (0.2 μM) whereas LM (200 μM) is much less effective.
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| 10. |
- Roberg, Karin, et al.
(författare)
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Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress
- 1999
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 27:11-12, s. 1228-1237
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Tidskriftsartikel (refereegranskat)abstract
- Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨm) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨm, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.
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