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What does the commonly used DCF test for oxidative stress really show?
Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences.
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2010 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 428, no 2, 183-90 p.Article in journal (Refereed) Published
Abstract [en]

H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

Place, publisher, year, edition, pages
2010. Vol. 428, no 2, 183-90 p.
Keyword [en]
age-related macular degeneration (AMD), 2', 7'-dichlorofluorescein (DCF), lysosome, oxidative stress, reactive oxygen species (ROS), transition metal.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-56466DOI: 10.1042/BJ20100208PubMedID: 20331437OAI: oai:DiVA.org:liu-56466DiVA: diva2:319384
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2017-12-12
In thesis
1. Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
Open this publication in new window or tab >>Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxidative stress is a major pathogenic factor in the development of age-related macular degeneration (AMD), which is the most common cause of severe central visual impairment in the elderly population in the western world.

It is believed that the degenerative process starts in the retinal pigment epithelium (RPE). The post-mitotic RPE is a single layer of pigmented cells located behind the photoreceptors – rods and cones – of the retina. Daily, the RPE cells phagocytose and recycle the expended tips of the photoreceptor outer segments. This heavy phagocytic burden leads to substantial oxidative stress in the cells, which is further enhanced by intense illumination and a high oxygen tension. A hallmark of early AMD is a progressive build-up of the non-degradable age pigment lipofuscin (LF) in lysosomes of the RPE. LF accumulation hampers phagocytosis and autophagy in the RPE, resulting in increased amounts of cellular debris in and around the cells. This decreases the function and viability of both RPE cells and photoreceptors.

Iron is known to accumulate in the retina with increasing age, particularly in AMDaffected eyes, and amplifies oxidative stress by acting as a potent catalyst in the generation of hydroxyl radicals. These highly reactive radicals contribute to LF formation and may, if abundantly present, also directly damage lysosomal membranes. The subsequent leakage of degrading enzymes to the cytosol initiates cell death via apoptosis or necrosis.

In this thesis, we have investigated the oxidative stress response of human RPE (ARPE-19) cells compared to murine J774 cells, another type of lysosome-rich cells with a high phagocytic capacity. The ARPE-19 cells were found to be extremely resistant to oxidative stress and tolerated exposure to single doses of H2O2 in concentrations up to 150 times higher than the J774 cells before lysosomal rupture and ensuing cell death occurred. This resistance was increased even further when the cells were protected with a potent iron chelator that prevents redox-active iron to participate in hydroxyl radical generation. Both cell lines were shown to be equally effective in degrading H2O2 and seem to contain comparable amounts of total as well as intralysosomal iron. Therefore, we reasoned that the insensitivity of ARPE-19 cells to H2O2 exposure might be related to a mechanism which keeps their intralysosomal iron bound in a non redox-active form. This theory was supported by our finding of very high basal expression levels of metallothionein (MT), heat shock-protein 70 (HSP70) and ferritin (FT) in ARPE-19 cells compared to J774 cells. All of these proteins have previously been shown to possess potent iron-binding properties. The ARPE-19 cells were also shown to have a higher basal rate of autophagy. SiRNA-mediated attenuation of MT, HSP70 and FT levels in the ARPE-19 cells resulted, to some degree, in an increased sensitivity to H2O2 treatment. Furthermore, a human cell stress array showed several other stress-related proteins to be up-regulated in ARPE-19 cells.

Additionally, we evaluated the commonly used, but frequently misinterpreted, H2DCF test for oxidative stress. It was demonstrated that oxidation of H2DCF into fluorescent DCF mainly reflects relocation to the cytosol of lysosomal iron and mitochondrial cytochrome c, rather than being the result of some poorly defined “general” oxidative stress.

In conclusion, our results indicate that the extreme resistance to oxidative stress exhibited by the ARPE-19 cells might be related to a high continuous autophagic influx of iron-binding proteins into the lysosomal compartment. Before being degraded, such proteins will temporarily keep intralysosomal iron bound in a non redox-active form, thereby inhibiting hydroxyl radical formation. This may partly explain why RPE cells, in spite of their exposed location and heavy burden of phagocytosis, usually manage to survive and evade significant LF accumulation until late in life.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 93 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1426
National Category
Clinical Medicine Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-111558 (URN)10.3384/diss.diva-111558 (DOI)978-91-7519-209-3 (ISBN)
Public defence
2014-11-28, Nils-Holgersalen, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
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Available from: 2014-10-24 Created: 2014-10-24 Last updated: 2015-09-29Bibliographically approved

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Karlsson, MarkusKurz, TinoBrunk, Ulf T.Nilsson, Sven E.Frennesson, Christina I.

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