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Wallace Eaton, John
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Publications (10 of 22) Show all publications
Gao, X., Li Campian, J., Qian, M., Sun, X.-F. & Wallace Eaton, J. (2009). Mitochondrial DNA Damage in Iron Overload. JOURNAL OF BIOLOGICAL CHEMISTRY, 284(8), 4767-4775
Open this publication in new window or tab >>Mitochondrial DNA Damage in Iron Overload
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2009 (English)In: JOURNAL OF BIOLOGICAL CHEMISTRY, ISSN 0021-9258, Vol. 284, no 8, p. 4767-4775Article in journal (Refereed) Published
Abstract [en]

Chronic iron overload has slow and insidious effects on heart, liver, and other organs. Because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological "memory." We hypothesized that cumulative iron-catalyzed oxidant damage to mtDNA might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. Real time PCR was used to examine the " intactness" of mttDNA in cultured H9c2 rat cardiac myocytes. After 3 -5 days exposure to high iron, these cells exhibited damage to mtDNA reflected by diminished amounts of near full-length 15.9-kb PCR product with no change in the amounts of a 16.1-kb product from a nuclear gene. With the loss of intact mtDNA, cellular respiration declined and mRNAs for three electron transport chain subunits and 16 S rRNA encoded by mtDNA decreased, whereas no decrements were found in four subunits encoded by nuclear DNA. To examine the importance of the interactions of iron with metabolically generated reactive oxygen species, we compared the toxic effects of iron in wild-type and rhoo cells. In wild-type cells, elevated iron caused increased production of reactive oxygen species, cytostasis, and cell death, whereas the rhoo cells were unaffected. We conclude that long-term damage to cells and organs in iron-overload disorders involves interactions between iron and mitochondrial reactive oxygen species resulting in cumulative damage to mtDNA, impaired synthesis of respiratory chain subunits, and respiratory dysfunction.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17151 (URN)10.1074/jbc.M806235200 (DOI)
Note
Original Publication:Xueshan Gao, Jian Li Campian, Mingwei Qian, Xiao-Feng Sun and John Wallace Eaton, Mitochondrial DNA Damage in Iron Overload, 2009, JOURNAL OF BIOLOGICAL CHEMISTRY, (284), 8, 4767-4775.http://dx.doi.org/10.1074/jbc.M806235200Copyright: American Society for Biochemistry and Molecular Biologyhttp://www.asbmb.org/Available from: 2009-03-12 Created: 2009-03-07 Last updated: 2009-08-18Bibliographically approved
Gao, X., Qian, M., Campian, J., Clark, D., Burke, T., Eaton, J. W. & McGregor, W. (2006). Cytotoxic and mutagenic effects of tobacco-borne free fatty acids. Free Radical Biology & Medicine, 40(1), 165-172
Open this publication in new window or tab >>Cytotoxic and mutagenic effects of tobacco-borne free fatty acids
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2006 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 40, no 1, p. 165-172Article in journal (Refereed) Published
Abstract [en]

Tobacco smoke contains substances capable of binding iron in an aqueous medium and transferring the metal into both organic solvents and intact mammalian red cells. This iron-binding activity is due to free fatty acids which are abundant in tobacco smoke and form 2:1 (free fatty acid:iron) chelates with ferrous iron. These earlier observations suggested that smoke-borne free fatty acids and the associated delocalization of iron within the lung might contribute to both the chronic pulmonary inflammation and the carcinogenesis associated with smoking. We now report that micromolar concentrations of iron or free fatty acid are not toxic to cultured human lung fibroblasts. However, when combined, the same low concentrations of iron and free fatty acid exert synergistic toxicity. Furthermore, the combination of free fatty acid and iron is highly mutagenic, inducing almost as many selectable mutations in the gene for hypoxanthine/guanine phosphoribosyl transferase as does benzo[a] pyrenediolepoxide, a class I carcinogen generated from benzo[a]pyrene present in cigarette smoke. The combination of free fatty acid and iron also promotes transformation of NIH 3T3 cells into an anchorage-independent phenotype. We conclude that free fatty acids in tobacco smoke may be important contributors to both the pulmonary damage and the carcinogenesis associated with smoking. © 2005 Elsevier Inc. All rights reserved.

Keywords
Cytotoxicity, Free fatty acids, Free radical, Iron, Mutagenesis, Tobacco
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-50270 (URN)10.1016/j.freeradbiomed.2005.09.033 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
Persson, H., Kurz, T., Eaton, J. W. & Brunk, U. (2005). Radiation-induced cell death: Importance of lysosomal destabilization. Biochemical Journal, 389(3), 877-884
Open this publication in new window or tab >>Radiation-induced cell death: Importance of lysosomal destabilization
2005 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 389, no 3, p. 877-884Article in journal (Refereed) Published
Abstract [en]

The mechanisms involved in radiation-induced cellular injury and death remain incompletely understood. In addition to the direct formation of highly reactive hydroxyl radicals (HO.) by radiolysis of water, oxidative stress events in the cytoplasm due to formation of H2O2 may also be important. Since the major pool of low-mass redox-active intracellular iron seems to reside within lysosomes, arising from the continuous intralysosomal autophagocytotic degradation of ferruginous materials, formation of H2O2 inside and outside these organelles may cause lysosomal labilization with release to the cytosol of lytic enzymes and low-mass iron. If of limited magnitude, such release may induce 'reparative autophagocytosis', causing additional accumulation of redox-active iron within the lysosomal compartment. We have used radio-resistant histiocytic lymphoma (J774) cells to assess the importance of intralysosomal iron and lysosomal rupture in radiation-induced cellular injury. We found that a 40 Gy radiation dose increased the 'loose' iron content of the (still viable) cells approx. 5-fold when assayed 24 h later. Cytochemical staining revealed that most redox-active iron was within the lysosomes. The increase of intralysosomal iron was associated with 'reparative autophagocytosis', and sensitized cells to Iysosomal rupture and consequent apoptotic/necrotic death following a second, much lower dose of radiation (20 Gy) 24 h after the first one. A high-molecular-mass derivative of desferrioxamine, which specifically localizes intralysosomally following endocytic uptake, added to the culture medium before either the first or the second dose of radiation, stabilized lysosomes and largely prevented cell death. These observations may provide a biological rationale for fractionated radiation. © 2005 Biochemical Society.

Keywords
Apoptosis, Ionizing radiation, Iron chelator, Lysosome, Macrophage, Oxidative stress
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-50449 (URN)10.1042/BJ20050271 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
Terman, A., Dalen, H., Eaton, J. W., Neuzil, J. & Brunka, U. (2004). Aging of cardiac myocytes in culture - Oxidative stress, lipofuscin accumulation, and mitochondrial turnover. Annals of the New York Academy of Sciences, 1019, 70-77
Open this publication in new window or tab >>Aging of cardiac myocytes in culture - Oxidative stress, lipofuscin accumulation, and mitochondrial turnover
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2004 (English)In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1019, p. 70-77Article in journal (Refereed) Published
Abstract [en]

Oxidative stress is believed to be an important contributor to aging, mainly affecting long-lived postmitotic cells such as cardiac myocytes and neurons. Aging cells accumulate functionally effete, often mutant and enlarged mitochondria, as well as an intralysosomal undegradable pigment, lipofuscin. To provide better insight into the role of oxidative stress, mitochondrial damage, and lipofuscinogenesis in postmitotic aging, we studied the relationship between these parameters in cultured neonatal rat cardiac myocytes. It was found that the content of lipofuscin, which varied drastically between cells, positively correlated with mitochondrial damage (evaluated by decreased innermembrane potential), as well as with the production of reactive oxygen species. These results suggest that both lipofuscin accumulation and mitochondrial damage have common underlying mechanisms, likely including imperfect autophagy and ensuing lysosomal degradation of oxidatively damaged mitochondria and other organelles. Increased size of mitochondria (possibly resulting from impaired fission due to oxidative damage to mitochondrial DNA, membranes, and proteins) also may interfere with mitochondrial turnover, leading to the appearance of so-called "giant" mitochondria. This assumption is based on our observation that pharmacological inhibition of autophagy with 3-methyladenine induced only moderate accumulation of large (senescent-like) mitochondria but drastically increased numbers of small, apparently normal mitochondria, reflecting their rapid turnover and suggesting that enlarged mitochondria are poorly autophagocytosed. Overall, our findings emphasize the importance of mitochondrial turnover in postmitotic aging and provide further support for the mitochondrial-lysosomal axis theory of aging.

Keywords
aging, heart, lipofuscin, lysosomes, mitochondria, oxidative stress
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-46195 (URN)10.1196/annals.1297.015 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
Li, W., Yuan, X., Ivanova, S., Tracey, K., Eaton, J. W. & Brunk, U. (2003). 3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin. Biochemical Journal, 371(2), 429-436
Open this publication in new window or tab >>3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin
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2003 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 371, no 2, p. 429-436Article in journal (Refereed) Published
Abstract [en]

Cytotoxic polyamine-derived amino aldehydes, formed during cerebral ischaemia, damage adjacent tissue (the so-called 'penumbra') not subject to the initial ischaemic insult. One such product is 3-aminopropanal (3-AP), a potent cytotoxin that accumulates in ischaemic brain, although the precise mechanisms responsible for its formation are still unclear. More relevant to the present investigations, the mechanisms by which such a small aldehydic compound might be cytotoxic are also not known, but we hypothesized that 3-AP, having the structure of a weak lysosomotropic base, might concentrate within lysosomes, making these organelles a probable focus of initial toxicity. Indeed, 3-AP leads to lysosomal rupture of D384 glioma cells, a process which clearly precedes caspase activation and apoptotic cell death. Immunohistochemistry reveals that 3-AP concentrates in the lysosomal compartment and prevention of this accumulation by the lysosomotropic base ammonia, NH3, protects against 3-AP cytotoxicity by increasing lysosomal pH. A thiol compound, N-(2-mercaptopropionyl)glycine, reacts with and neutralizes 3-AP and significantly inhibits cytoxocity. Both amino and aldehyde functions of 3-AP are necessary for toxicity: the amino group confers lysosomotropism and the aldehyde is important for additional, presently unknown, reactions. We conclude that 3-AP exerts its toxic effects by accumulating intralysosomally, causing rupture of these organelles and releasing lysosomal enzymes which initiate caspase activation and apoptosis (or necrosis if the lysosomal rupture is extensive). These results may have implications for the development of new therapeutics designed to lessen secondary damage arising from focal cerebral ischaemia.

Keywords
3-Aminopropanal, Amine oxidase, Apoptosis, Cerebral ischaemia, Lysosome
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-46662 (URN)10.1042/BJ20021520 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
Yu, Z., Persson, L., Eaton, J. W. & Brunk, U. (2003). Intralysosomal iron: a major determinant of oxidant-induced cell death. Free Radical Biology & Medicine, 34(10), 1243-1252
Open this publication in new window or tab >>Intralysosomal iron: a major determinant of oxidant-induced cell death
2003 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 34, no 10, p. 1243-1252Article in journal (Refereed) Published
Abstract [en]

As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH4Cl). The consequent increase in lysosomal pH (from ca. 4.5 to > 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH4Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H2O2, and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH4Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H2O2 addition, was absent in cells preexposed to 10 mM NH4Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27102 (URN)10.1016/S0891-5849(03)00109-6 (DOI)11749 (Local ID)11749 (Archive number)11749 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
Zhao, M., Antunes, F., Eaton, J. W. & Brunk, U. (2003). Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis. European Journal of Biochemistry, 270(18), 3778-3786
Open this publication in new window or tab >>Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis
2003 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 18, p. 3778-3786Article in journal (Refereed) Published
Abstract [en]

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant proapoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27712 (URN)10.1046/j.1432-1033.2003.03765.x (DOI)12450 (Local ID)12450 (Archive number)12450 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
Brunk, U., Yu, Z., Persson, L. & Eaton, J. W. (2003). Lysosomes, iron and oxidative stress. Free radical research, 37
Open this publication in new window or tab >>Lysosomes, iron and oxidative stress
2003 (English)In: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 37, p. 34-34Conference paper, Published paper (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-48512 (URN)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
Terman, A., Dalen, H., Eaton, J. W., Neuzil, J. & Brunk, U. (2003). Mitochondrial recycling and aging of cardiac myocytes: The role of autophagocytosis. Experimental Gerontology, 38(8), 863-876
Open this publication in new window or tab >>Mitochondrial recycling and aging of cardiac myocytes: The role of autophagocytosis
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2003 (English)In: Experimental Gerontology, ISSN 0531-5565, E-ISSN 1873-6815, Vol. 38, no 8, p. 863-876Article in journal (Refereed) Published
Abstract [en]

The mechanisms of mitochondrial alterations in aged post-mitotic cells, including formation of so-called 'giant' mitochondria, are poorly understood. To test whether these large mitochondria might appear due to imperfect autophagic mitochondrial turnover, we inhibited autophagocytosis in cultured neonatal rat cardiac myocytes with 3-methyladenine. This resulted in abnormal accumulation of mitochondria within myocytes, loss of contractility, and reduced survival time in culture. Unlike normal aging, which is associated with slow accumulation of predominantly large defective mitochondria, pharmacological inhibition of autophagy caused only moderate accumulation of large (senescent-like) mitochondria but dramatically enhanced the numbers of small mitochondria, probably reflecting their normally more rapid turnover. Furthermore, the 3-methyladenine-induced accumulation of large mitochondria was irreversible, while small mitochondria gradually decreased in number after withdrawal of the drug. We, therefore, tentatively conclude that large mitochondria selectively accumulate in aging post-mitotic cells because they are poorly autophagocytosed. Mitochondrial enlargement may result from impaired fission, a possibility supported by depressed DNA synthesis in large mitochondria. Nevertheless, enlarged mitochondria retained immunoreactivity for cytochrome c oxidase subunit 1, implying that mitochondrial genes remain active in defective mitochondria. Our findings suggest that imperfect autophagic recycling of these critical organelles may underlie the progressive mitochondrial damage, which characterizes aging post-mitotic cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27710 (URN)10.1016/S0531-5565(03)00114-1 (DOI)12448 (Local ID)12448 (Archive number)12448 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
Persson, L., Yu, Z., Tirosh, O., Eaton, J. W. & Brunk, U. (2003). Prevention of oxidant-induced cell death by lysosomotropic iron chelators. Free Radical Biology & Medicine, 34(10), 1295-1305
Open this publication in new window or tab >>Prevention of oxidant-induced cell death by lysosomotropic iron chelators
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2003 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 34, no 10, p. 1295-1305Article 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27105 (URN)10.1016/S0891-5849(03)00106-0 (DOI)11752 (Local ID)11752 (Archive number)11752 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
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