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The radioprotective agent, amifostine, suppresses the reactivity of intralysosomal iron
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Faculty of Health Sciences.
James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Department of Medicine and Care, Pulmonary Medicine. Linköping University, Faculty of Health Sciences.
2003 (English)In: Redox report, ISSN 1351-0002, E-ISSN 1743-2928, Vol. 8, no 6, 347-355 p.Article in journal (Refereed) Published
Abstract [en]

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 ?M WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.

Place, publisher, year, edition, pages
2003. Vol. 8, no 6, 347-355 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27104DOI: 10.1179/135100003225003384Local ID: 11751OAI: oai:DiVA.org:liu-27104DiVA: diva2:247655
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
In thesis
1. 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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