Detection and Characterization of the Electron Paramagnetic Resonance-Silent Glutathionyl-5,5-dimethyl-1-pyrrolineN-Oxide Adduct Derived from Redox Cycling of Phenoxyl Radicals in Model Systems and HL-60 Cells

Stoyanovsky, Detcho A.; Goldman, Radoslav; Jonnalagadda, Sastry S.; Day, Billy W.; Claycamp, H. Gregg; Kagan, Valerian E.

doi:10.1006/abbi.1996.0219

Cited by 48 publications

(50 citation statements)

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“…Peroxynitrite is one of the biological oxidants whose addition to cells and cell cultures has been shown to either activate signaling pathways (25)(26)(27) or lead to cell injury (28 -30, 50), depending on cell type and oxidant concentration. Both of these cellular processes have been associated with protein-thiol oxidation, which in general is controlled by the intracellular concentration of low molecular weight thiols such as glutathione in the case of mammalian cells (51)(52)(53)(54). In this context, our results demonstrating peroxynitrite-mediated oxidation of intracellular glutathione to glutathionyl radicals may be relevant to the understanding of the bioregulatory and biodamaging roles of the oxidant.…”

Section: Bmentioning

confidence: 88%

EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774)

Menezes

Augusto

2001

Journal of Biological Chemistry

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Peroxynitrite is one of the biological oxidants whose addition to cells has been shown to either activate signaling pathways or lead to cell injury, depending on cell type and oxidant concentration. The intermediacy of free radicals in these processes has been directly demonstrated only during the interaction of peroxynitrite with erythrocytes, a particular cell type, due to its high hemoglobin content. Here, we demonstrate that the addition of peroxynitrite to a macrophage cell line (J774) led to the production of glutathionyl and protein-tyrosyl radicals. The glutathionyl radical was characterized by EPR spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide. Protein-tyrosyl radical formation was suggested by direct EPR spectroscopy and confirmed by EPR spin-trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid and Western blot analysis of nitrated proteins in treated macrophages. Time dependence studies of free radical formation indicate that intracellular glutathione and unidentified proteins are the initial peroxynitrite targets in macrophages and that their derived radicals trigger radical chain reactions. The results are likely to be relevant to the understanding of the bioregulatory and biodamaging effects of peroxynitrite.Peroxynitrite (ONOO Ϫ ϩ ONOOH), 1 which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of human diseases and as a toxin against invading microorganisms (1-5). The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that are presently being elucidated (6 -16). Peroxynitrite-mediated oxidations are either bimolecular, first-order on peroxynitrite and target concentration or unimolecular, first order on peroxynitrite and independent of target concentration. Bimolecular processes can result in product yield either around stoichiometry and above, as is the case for thiol oxidation (17), or around 35%, as is the case for carbon dioxide oxidation (10, 14 -16). Presently, most investigators accept that product yields around 30% are characteristic of peroxynitrite-mediated free radical processes. Indeed, it has been established that peroxynitrite protonation (pK a ϭ 6.6) leads to its fast decomposition (k ϭ 0.17 s Ϫ1 and t1 ⁄2 ϭ 4.1 s at pH 7.4, 25°C) to yield ϳ70% nitrate and 30% hydroxyl radical and nitrogen dioxide (Reactions 1 and 2) (7-9, 11-14). These radicals are the species responsible for peroxynitrite-mediated unimolecular oxidations.In biological systems, the half-life of peroxynitrite is expected to be much lower because of its reactions with biotargets, particularly carbon dioxide, hemoproteins, and thiol-containing compounds, all of which react fast with the oxidant in bimolecular reactions (2,18,19). The reaction with the biologically ubiquitous carbon dioxide is particularly fast (k ϭ 2.6 ϫ 10 4 M Ϫ1 s Ϫ1 at pH 7.4, 25°C) and produces ϳ65% nitrate and 35% carbonate radical anion and nitrogen dioxide (Reaction 3) (10, 14 -16, 1...

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Section: Bmentioning

confidence: 88%

EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774)

Menezes

Augusto

2001

Journal of Biological Chemistry

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“…It is possible to initiate this oxidation through a process referred to as "thiol pumping" in which a compound, typically a phenol, is metabolized to a reactive free radical by one of a number of peroxidase enzymes, and the radicals so formed are reduced by a thiol, forming a very reactive thiyl radical (29 -36). Thiyl and superoxide radicals produced in this manner from glutathione have been observed by spin trapping (29,31,33,34) while the glutathione disulfide radical anion has been observed directly with ESR (30).Investigators have, until now, been unable to detect sulfurcentered radicals from dihydrolipoic acid in such a system (31, 33) although superoxide production and cycling of the phenoxyl radical back to phenol indicate that the dihydrolipoic acid is being oxidized. There was also a failure to detect the thiyl radical when dihydrolipoic acid reduced met-myoglobin to oxymyoglobin (37), a situation in which one would expect the radical to be formed.…”

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confidence: 99%

Sulfur-centered Radical Formation from the Antioxidant Dihydrolipoic Acid

Mottley¹,

Mason²

2001

Journal of Biological Chemistry

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Lipoic acid and its reduced form, dihydrolipoic acid, are thought to be strong antioxidants. There are also reports of dihydrolipoic acid acting as a pro-oxidant under certain circumstances. This article reports the direct observation by ESR spectrometry of the disulfide radical anion and the spin trapping of the primary thiyl radical formed from the oxidation of dihydrolipoic acid through thiol pumping with phenol and horseradish peroxidase. The disulfide radical anion reacts rapidly with oxygen to form the reactive radical superoxide, which is also trapped. The radical species formed show a potential for pro-oxidant activity of this compound. Although antioxidants, in general, have been shown to have pro-oxidant potential, the pro-oxidant chemistry of dihydrolipoic acid has not been well characterized.There has been much recent interest in ␣-lipoic acid as a powerful antioxidant in animals based primarily on observations that it or its reduced form, dihydrolipoic acid, scavenges hydroxyl radical (1-3), superoxide (2), singlet oxygen (4, 5), and hypochlorous acid (1, 6, 7). In addition, lipoic acid recycles the well known antioxidants ascorbate, glutathione, and vitamin E by reducing their radical form back to the parent compound (8 -11). The antioxidant properties have been reviewed in several papers (12, 13) and so will not be extensively discussed here. Lipoic acid is used to treat toxicities in which free radicalinduced peroxidation of membrane phospholipids is thought to be important (14 -16) and, with vitamin E, has been shown to improve cardiac function after ischemia in rats (17).On the other hand, there are a limited number of reports describing potential pro-oxidant effects of dihydrolipoic acid such as releasing iron from ferritin (18), reducing Fe 3ϩ to Fe 2ϩ in microsomes (19), accelerating deoxyribose degradation by FeCl 3 -EDTA and H 2 O 2 (1), peroxidation of oxidized brain phospholipid liposomes in the presence of Fe 3ϩ (1), accelerating ␣ 1 -antiproteinase inactivation (1), and accelerating the loss of creatine kinase activity in plasma exposed to gas-phase cigarette smoke (1).The oxidation of thiols involves quite complicated chemistry and a number of free radical species (20 -28), each of which has the potential for causing damage in a biological system. It is possible to initiate this oxidation through a process referred to as "thiol pumping" in which a compound, typically a phenol, is metabolized to a reactive free radical by one of a number of peroxidase enzymes, and the radicals so formed are reduced by a thiol, forming a very reactive thiyl radical (29 -36). Thiyl and superoxide radicals produced in this manner from glutathione have been observed by spin trapping (29,31,33,34) while the glutathione disulfide radical anion has been observed directly with ESR (30).Investigators have, until now, been unable to detect sulfurcentered radicals from dihydrolipoic acid in such a system (31, 33) although superoxide production and cycling of the phenoxyl radical back to phenol indicate that the d...

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“…This increased GSH turnover, as evidenced by increased formation of the glutathionyl radical, is not related to changes in GSH peroxidase activity, as such changes are not associated with glutathionyl radical formation (Stoyanovsky et al, 1996). They are rather related to the non-enzymatic generation of glutathionyl radical via interaction with NCS (Stoyanovsky et al, 1996;. This finding, along with our previous observation that the downstream block in the apoptosis final common pathway produced by Bcl-2 is lifted in NCS-treated cells , explains the enhanced apoptotic rate in bcl-2-transfected PC 12 cells treated with the reduction-dependent prodrug, NCS.…”

Section: Percent Of Total Phospholipidsmentioning

confidence: 86%

“…In addition, our finding that the rate of turnover of GSH is higher in Bcl-2-overproducing PC 12 cells than in native producers exposed to NCS implies that not only the endogenous rate of production of ROS, but also the role of GSH in and ability to compensate for consumption of reducing equivalents is aberrant in some bcl-2-overexpressing cells. This increased GSH turnover, as evidenced by increased formation of the glutathionyl radical, is not related to changes in GSH peroxidase activity, as such changes are not associated with glutathionyl radical formation (Stoyanovsky et al, 1996). They are rather related to the non-enzymatic generation of glutathionyl radical via interaction with NCS (Stoyanovsky et al, 1996;.…”

Section: Percent Of Total Phospholipidsmentioning

confidence: 89%

“…Effect of NCS on formation of glutathionyl radical: Mock-and bcl-2-transfected PC 12 cells treated with varying concentrations of NCS were assayed for formation of the glutathionyl radical as a measure of glutathione turnover using the free radical trapping agent DMPO as we have previously described (Stoyanovsky et al, 1996). Briefly, cells…”

Section: Percent Of Total Phospholipidsmentioning

confidence: 99%

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Exploiting BCL-2 Overexpression in the Chemotherapy of Breast Cancer

Schor¹

1998

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project 10704-0188), Washington, DC 20503. AGENCY USE ONLY (Leave blank)2. REPORT DATE June 19994. TITLE AND SUBTITLE REPORT TYPE AND DATES COVERED Annual_(l Jun 98 -31 May FUNDING NUMBERS 99:Exploiting bcl-2 Overexpression in the Chemotherapy of Breast Cancer AUTHOR(S)Schor, Nina F., M.D., Ph.D. DAMD17-97-1-7247 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Children's Hospital of Pittsburgh Pittsburgh, Pennsylvania 152138. PERFORMING ORGANIZATION REPORT NUMBER In neural crest cells, bcl-2 overexpression results in altered handling of glutathione, and consequent increased cellular reducing potential. This change potentiates the induction of apoptosis by enediyne prodrugs in neural crest tumor cells. Our studies examined the applicability of these findings to MCF-7 breast cancer cells, known to overexpress bcl-2 in response to estradiol treatment. We have found that estradiol treatment of MCF-7 cells does indeed result in an increase in the expression of bcl-2. However, transfection of MCF-7 cells with an expression construct for bcl-2 does not result in potentiation of enediyne-induced apoptosis. Unlike the case for neural crest tumor cells, glutathione handling is not altered in MCF-7 cells induced by transfection to overexpress bcl-2. Our results to date suggest that determination of the effect of bcl-2 expression on glutathione handling may allow prediction of the response of a particular tumor cell to treatment with enediynes. In the one-year extension period of this grant, we will merge these two lines of investigation and determine the effects of estradiol treatment (rather than direct bcl-2 transfection) on glutathione handling in MCF-7 cells. Initial studies indicate that estradiol treatment does alter the native glutathione content of MCF-7 cells. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U14. SUBJECT Where copyrighted material is quoted, permission has been obtained to use such material.Where material from documents designated for limited distribution is quoted, permission has been obtained to use the material.Citations of commercial organizations and trade names in this report do not constitute an official Department of Army endorsement or approval of the products or services of these organizations.In conducting research using animals, the investigator(s) adhered to the "Guide for the Care and Use of Laboratory Animals," prepared by the Commit...

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Detection and Characterization of the Electron Paramagnetic Resonance-Silent Glutathionyl-5,5-dimethyl-1-pyrrolineN-Oxide Adduct Derived from Redox Cycling of Phenoxyl Radicals in Model Systems and HL-60 Cells

Cited by 48 publications

References 0 publications

EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774)

EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774)

Sulfur-centered Radical Formation from the Antioxidant Dihydrolipoic Acid

Exploiting BCL-2 Overexpression in the Chemotherapy of Breast Cancer

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