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Prevention of oxidant-induced cell death by lysosomotropic iron chelators
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Department of Medicine and Care, Pulmonary Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Faculty of Health Sciences.
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
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2003 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 34, no 10, 1295-1305 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2003. Vol. 34, no 10, 1295-1305 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27105DOI: 10.1016/S0891-5849(03)00106-0Local ID: 11752OAI: oai:DiVA.org:liu-27105DiVA: diva2:247656
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-16Bibliographically approved
In thesis
1. Prevention of oxidant-induced cell death by intralysosomal iron binding
Open this publication in new window or tab >>Prevention of oxidant-induced cell death by intralysosomal iron binding
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The lung is particularly prone to oxidative stress by its exposure to ambient oxygen and inhaled environmental oxidants. Abnormal assimilation and accumulation of iron are found in many lung disorders, which in redox-active form will exacerbate oxidative tissue damage. It may be that the most important cellular pool of redox -active iron exists within lysosomes. As a result, these organelles are very vulnerable to oxidative stress and may burst due to peroxidative membrane destabilization. Support for the importance of intralysosomal iron in cellular oxidant damage includes the observation that the iron chelator, desferrioxamine, which almost exclusively localizes within the lysosomal compartment, will protect cells against oxidati ve challenge. Iron chelators targeted to the lysosomes may therefore be a particularly efficient therapeutic strategy for cells under conditions of substantial oxidative stress.

The present study, employing cultures of human respiratory epithelial cells and murine macrophage-like cells, explores the protective effects by iron binding agents upon H202 and gamma radiation-induced lysosomal damage and cell death. Using these in vitro models, the present study shows: (1) that chelation of intralysosomal iron efficiently prevents lysosomal rupture and ensuing cell death induced by either H202 or gamma radiation; (2) that cell permeable lysosomotropic iron-chelators are much more efficient than those being internalized by endocytosis; (3) that intralysosomal iron is the most important cellular pool of redox-active iron for chelation therapy; (4) that ironcatalyzed peroxidative lysosomal destabilization is a decisive and early event in the apoptotic machinery.

Although apoferritin and desferrioxarnine suppress the reactivity of lysosomal iron, their efficacy is considerably restrained by their uptake by fluid-phase endocytosis. Apoferritin is digested intralysosomally which further decreases its iron sequestering potential, while desferrioxamine by its intralysosomal retention may disturbe normal cellular functions and cause iron-starvation. Amongst cell permeable iron-binding agents we tested a-lipoic acid, alipoamide, and a synthetic amine derivative of α-lipoarnide, α-lipoic acid-plus (5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl) amide). The large difference in the protective potential of these cell permeant iron-chelators derives from their being localized in different cellular compartments, which lends further support that lysososomes contain the most important pool of chelatable redox-active iron. Indeed, a-lipoic acid-plus by its lysosomotropism was by all means the most efficient iron chelator. On a molar basis α-lipoic acid-plus was 4,000 to 5,000 times more effective than desferrioxamine to prevent lysosomal rupture and cell death induced by H202 or gamma radiation.

We conclude that iron chelating therapy targeted to the lysosomes is an efficient strategy to protect oxidatively stressed cells in vitro. A corresponding efficacy of such treatment in vivo, and in iron dependent pulmonary disorders in particular, needs to be explored.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 60 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 812
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27537 (URN)12194 (Local ID)91-7373-502-7 (ISBN)12194 (Archive number)12194 (OAI)
Public defence
2003-10-23, Victoriasalen, Universitetssjukhuset, Linköping, 13:15 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-05Bibliographically approved
2. Proton trapping in the cellular acidic vacuolar compartment: lysosomal mechanisms in apoptosis/necrosis and iron chelation
Open this publication in new window or tab >>Proton trapping in the cellular acidic vacuolar compartment: lysosomal mechanisms in apoptosis/necrosis and iron chelation
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Under ischemic conditions, a number of cytotoxic metabolic products are formed. Reactive oxygen species are known to be important mediators of progressive ischemic cell injury, and the synergistic damage to cells caused by the combination of such oxygen species and redox-active iron is well appreciated. The acidic interior of lysosome leads to the trapping of substances with high pK4 values. A large variety of molecules, being weak bases, may thus concentrate within this acidic vacuolar compartment, potentially leading to both beneficial and detrimental effects. A major part of the intracellular pool of redoxactive iron is likely to be located in the lysosomal compartment, and iron chelators that are lysosomotropic due to high pK4 values may prove to be important pharmacological tools to protect the brain from oxidative stress. Among a variety of substances formed in the ischemic penumbra zone is the polyamine metabolite, 3-aminopropanal (3-AP), a substance of extreme neurotoxicity. 3-AP is a weak base and may theoretically exert its toxic action through induction of cell death after intralysosomal accumulation.

On the 1774 mouse histiocytic lymphoma cell line, we used the common lysosomotropic agent NH3 to increase lysosomal pH, the lysosomotropic iron chelator, 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (LAP) and the lysosomotropic iron binder, WR-1065, a metabolite of amifostine, as tools to determine that proton trapping within the lysosomal acidic vacuolar compartment plays an important role in oxidative stress-induced apoptosis. We also used another lysosomotropic agent, 3-AP, on the J774 cell line and on the SH-SY5Y human neuroblastoma cell line. The results indicate that proton trapping of this toxin within the lysosome might explain its toxicity to cells.

Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in the lysosomal content of redox-active iron following reduced acidity of the lysosome, and electron spin-resonance studies showed that no hydroxyl radicals [OH] were formed from hydrogen peroxide under these conditions. This suggests that lysosomes contain most of the free, redox-active iron. In further support of this idea, the lysosomotropic agents LAP and WR-1065 were found to be 5000 and 2500 times more effective, respectively, in protecting cells from oxidative stress, compared with the well-known iron chelator desferrioxamine [DFO]. Evidence was obtained that LAP and WR-1065 exerted their effect on intralysosomal redox-active iron, and that the effect was linked to the acidity of the lysosome. Being weak bases (LAP, pKa = 8.0; WR-1065, pKa = 9.2), these compounds accumulate intralysosomally by proton trapping. The neurotoxic effect of 3-AP (pKa = 9.3) could be linked to a dose-dependent induction of cell death, most likely based on intralysosomal proton trapping of this molecule followed by lysosomal rupture. The lysosomal rupture seems to induce a chain of intracellular events (including generation of oxidative stress), leading to mitochondrial damage directly or indirectly caused by the release of lysosomal proteases.

We conclude that the low pH of the lysosome may both serve to attract basic toxins, such as 3-AP, and promote the accumulation of protective agents, such as LAP and WR-1065. Prevention of lysosomal damage from both oxidants and neurotoxins by lysosomotropic agents has great potential therapeutic utility.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 808
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28088 (URN)12854 (Local ID)91-7373-501-9 (ISBN)12854 (Archive number)12854 (OAI)
Public defence
2003-10-09, Patologens föreläsningssal, Universitetssjukhuset, Linköping, 13:15 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-16Bibliographically approved

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Persson, LennartYu, ZhengquanEaton, John WallaceBrunk, Ulf

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