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4-Hydroxy-2-Nonenal Mediates Aifm2 Release From Mitochondria: An Insight Into the Mechanism of Oxidative Stress Mediated Retrograde Signaling
sensitive labeling assays predict the presence of a redoxsensitive cysteine in a-ENaC sequence emphasizing the importance of sulfhydryl bonds for channel activity. These results promote a model in which ROS modulates the activity of the ENaC by promoting oxidation of regulatory sulfhydryls.
doi : 10.1016/j.freeradbiomed.2011.10.071
48 Cell-Based Analysis of Lipid Peroxidation and Lipid Peroxidation-Derived Protein Modifications Using Fluorescence Microscopy and High Content Imaging Bhaskar S Mandavilli1, Robert J Aggeler1, Aleksey Rukavishnikov1, Chad Pickens1, Upinder Singh1, Hee Chol Kang1, Kyle Gee1, Brian Agnew1, and Michael S Janes1 1 Life Technologies Corporation’ Eugene, OR, USA Oxidative stress plays an important role in the progression of several diseases including inflammation, atherosclerosis, aging and age-related degenerative disorders. Reactive oxygen species damage membrane bound lipids including unsaturated fatty acids like linoleic acid and arachidonic acid to form lipid electrophiles, which can rapidly react with proteins and DNA to form adducts. Cell-based measurements of lipid peroxidation and protein carbonylation by traditional fluorescence microscopy and high content imaging provide a powerful platform to quantitate lipid peroxidation in cells and also to monitor spatial distribution of damage caused by lipid peroxides. Here, we used two different approaches to measure lipid peroxidation in cells. 1) a ratiometric determination of lipid peroxidation in live cells using a fluorescent probe which is incorporated into cellular membranes and emits at 590 nm. Oxidation of the dye resulted in a fluorescence shift to a peak emission of 510 nm. the ratio of 590/510 nm emissions can be quantitated by traditional fluorescence microscopy and high content imaging, 2) in a click chemistry-based approach, alkyneor azide-modified unsaturated fatty acid analogs like linoleic acid or arachidonic acid were incorporated into the cellular membranes and the products resulting from oxidation, like 4-hydroxy-2nonenal (HNE) and 9, 12-dioxo-10(E) dodecenoic acid (DODE) can readily modify DNA or proteins. the modified proteins are then detected by a copper-catalyzed click reaction using fluorescent alkynes or azides. Using these approaches, we measured lipid peroxidation caused by oxidants like tert-butyl hydroperoxide (TBHP), cumene hydroperoxide (CH), menadione, hemin and lipolysaccharide (LPS) in bovine pulmonary endothelial cells and murine macrophage-like cells. the oxidants produced 2-3 fold increases in lipid peroxidation and protein modifications when compared to controls. the lipid peroxidation and the lipid peroxidederived protein modifications were successfully inhibited by using antioxidants like α-tocopherol and mixed tocopherols. the two strategies described here to measure lipid peroxidation and the derived protein modifications provide powerful tools to measure oxidative stress in cells.
doi : 10.1016/j.freeradbiomed.2011.10.072
S30
49 Inactivation of α-Ketoglutarate Dehydrogenase by 4Hydroxy-2-nonenal; Modification and Metabolic Fate of the Cofactor Lipoic Acid Aaron L. McLain1,2, Kenneth M. Humphries1,2, Daniel Stofan1, Michael T. Kinter1, and Luke I. Szweda1,2 1 Free Radical Biology and Aging Program, Oklahoma Medical 2 Research Foundation, Department of Biochemistry, University of Oklahoma Health Sciences Center The mitochondrial electron transport chain is a source of oxygen derived free radicals and pro-oxidants. Polyunsaturated fatty acids present within the mitochondrial membrane are in close proximity and are highly susceptible to oxidation. Lipid peroxidation can disrupt mitochondrial function by compromising membrane integrity and through the formation of reactive aldehydes, alkenals, and hydroxyalkenals. We previously demonstrated that the lipid peroxidation product 4-hydroxy-2nonenal (HNE) readily inactivates α-ketoglutarate dehydrogenase upon modification of the covalently bound cofactor lipoic acid. This results in reductions in the rates of NADH production and oxidative phosphorylation. Results of the present study provide evidence that, of the various lipid peroxidation products, hydroxyalkenals are the most potent inactivators of αketoglutarate dehydrogenase. Because HNE is the most abundant hydroxyalkenal formed during lipid peroxidation, antibodies were prepared that are specific to the HNE-lipoic acid adduct, exhibiting no cross reactivity to adducts formed between lipoic acid and structurally related products. These antibodies were used to demonstrate modification of lipoic acid on the E2 subunit of α-ketoglutarate dehydrogenase upon treatment of mitochondria with HNE. Once formed, the HNE-lipoic acid adduct disappeared over time while the level of the E2 subunit remained unchanged. Evidence indicates that the lipoic acid-HNE adduct is removed from the protein as the first step in a repair process.
doi : 10.1016/j.freeradbiomed.2011.10.073
50 4-Hydroxy-2-Nonenal Mediates Aifm2 Release From Mitochondria: An Insight Into the Mechanism of Oxidative Stress Mediated Retrograde Signaling Sumitra Miriyala1, Chadinee Thipakkorn2, Yong Xu1, Teresa Noel1, Craig Vander Kooi1, Ines Batinic-Haberle3, Virapong Prachayasittikul2, and Daret St Clair1 1 2 3 University of Kentucky, Mahidol University, Duke University Mitochondria, in addition to generation of ATP and production of ROS, have a critical role in signaling events leading to apoptosis or survival under normal and pathophysiological conditions. However, it is unknown how ROS generated in mitochondria signals the communication between mitochondria and the nucleus under life and death conditions. Here, we report the identification of the mitochondrion-associated apoptotic protein 2 (AIFm2), a p53 target gene, as the messenger for ROS-mediated retrograde signaling under stress conditions. AIFm2 is a flavoprotein of relative molecular mass 41,000, which possesses NAD(P)H oxidase activity and catalyzes NAD(P)H dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. in vivo, treatment with Doxorubicin (Dox), a ROS generating chemotherapeutic drug, significantly enhanced the levels of 4-hydroxy-2-nonenal (4HNE) and AIFm2 protein in cardiac tissues. Using immunoprecipitation and immunofluorescence we found that resting AIFm2 localized to mitochondria, whereas HNE-adducted AIFm2 localized to nucleus suggesting that HNE modification activates the shuttling of AIFm2. When purified AIFm2 was exposed to 4HNE in vitro,
SFRBM 2011
4HNE selectively modified His174 and Cys187 of AIFm2 assessed by mass spectroscopic analysis. Lentivirus expressed wild-type and C187T mutant AIFm2 migrated from mitochondria to nucleus upon exposure to Dox which is prevented by addition of the MnSOD mimetic whereas the H174R mutant protein remained in the mitochondria after exposure to Dox. the HNE-adducted AIFm2 exhibited strong DNA binding affinity whereas the H174R mutant demonstrated no DNA binding capability. the results demonstrate that AIFm2 is a redox-sensitive protein and that its translocation is controlled by mitochondrial ROS, which may explain the apoptosis function associated with AIFm2 as a p53 target gene.
do i : 10.1016/j.freeradbiomed.2011.10.074
51 Structural Characterization of Oxidation Susceptible Cysteines: a Step Towards Understanding Hydrogen Peroxide Mediated Signaling Santhosh Kannan ` Venkatesan1, Boopathy Rathanam1, and Lakshmi Venkatachalam1 1 Department of Biotechnology, Bharathiar University, Coimbatore, India Protein thiols particularly redox regulatory cys are the primary and direct targets of H2O2 oxidation which in turn regulate H2O2 mediated signaling. the susceptibility of a cys is influenced by its physico-chemical parameters which are in turn governed by their site of distribution. Neither structural nor sequence determinants have been reported for H2O2 oxidizable cys. Hence, spatial and structural restraints that influence the oxidation of cys by H2O2 is a pre-requisite for the identification of target proteins. An attempt has been made to study the structural parameters that distinguish the susceptible cys from that of non-susceptible ones. A database of known H2O2 oxidizable (structural protein and six different classes of enzyme) proteins was generated. It consists of 64 susceptible cys residues and 70 non-susceptible ones. the parameters considered for identification of H2O2 susceptible cys were i) the pKa of target cys ii) distance to the adjacent cys S (S1) iii) solvent accessibility of S1 and iv) distance to the nearby acidic and basic amino acids. The pKa of most susceptible cys was found to be less than 8.5, while that of non-susceptible cys was above 9. When the pKa of susceptible cys is above the cut-off range of 8.5, the deprotonation of such target cys was achieved by S1 or by the adjacent acidic amino acid. They help in its maintenance as thiolate anion. the other susceptible cys residues whose pKa value is lower than 8.5 was observed only with oxido-reductases enzymes. the second determinant is the distance of S1 to the target cys. If it is at or below 6Å, it normally favored the target cys to its anionic form. a distance below 3Å resulted in permanent disulphide linkage thereby reducing the susceptibility to H2O2. So the pKa of target cys and distance of S1 to target cys were the major discriminating parameters that differentiate susceptible cys from non-susceptible ones. There were exceptions too, which were compensated by the increased solvent accessible area of the S1 and its proximity to acidic aminoacid. the validity of these parameters and their applicability were cross checked with two proteins which are known targets of H2O2. These parameters identified will help to develop an algorithm to predict target proteins for H2O2 signaling.
doi : 10.1016/j.freeradbiomed.2011.10.075
52 Carbonylation and Inhibition of Mitochondrial Complex I during Kainate-Induced Epileptogenesis Kristen R Ryan1, Li-Ping Liang1, Donald S Backos1, Philip Reigan1, and Manisha Patel1 1 University of Colorado Denver, Department of Pharmaceutical Sciences Nearly one percent of the population suffers from epilepsy and acquired epilepsies such as temporal lobe epilepsy (TLE) resulting from head trauma, chemical exposure or infections. Like many progressive disorders, mitochondrial dysfunction and oxidative stress are linked to the development of TLE. a single injection of kainate, a glutamatergic agonist, results in acute excitotoxicity and chronic development of spontaneous seizures via a process termed epileptogenesis. We have previously shown increased mitochondrial-specific reactive oxygen species (ROS) production, mitochondrial DNA damage and decreases in mitochondrial redox status in the hippocampus of a rat kainate model of epileptogenesis (Jarrett S et al 2008; Liang L et al 2006). Increasing evidence suggests that ROS-induced protein posttranslational modifications are a contributing factor to mitochondrial dysfunction. We hypothesize that Complex I (CI) dysfunction may be a major cause of mitochondrial dysfunction and contribute to epileptogenesis. the goal of this study was to determine if oxidative modification of the 75kDa subunit (Ndufs1) of CI occurs in the rat hippocampus during epileptogenesis and whether this correlates with disease progression. Rats were injected with a single high dose of kainate or vehicle and monitored by video for seizure activity for 6 weeks. Mitochondrial CI activity was significantly decreased acutely (8h-2d) and chronically (6wk) after kainate injection; whereas ATP levels were persistently decreased throughout the 6 week period. Evidence for CI modifications was measured acutely after kainate administration, days after kainate prior to development of epilepsy (i.e. latent period) and during the chronic stages of epilepsy. CI was immunoprecipitated (Mitosciences, OR) from hippocampal tissue of vehicle and kainate treated rats and subunits were separated by SDS-PAGE. Ndufs1 modification was identified with an Agilent 6510 QTOF LC/MS system and MS/MS peptide data was dually analyzed with Mascot 2.2 (Matrix Science) and SpectraMill (Agilent). a correlation was observed between total mitochondrial carbonylation, specific Ndufs1 carbonylation at Arginine 76 (Arg76), and inhibition of CI activity throughout the course of epileptogenesis. Molecular modeling (Accelrys Discovery Studio 3.0) studies revealed that Arg76 is not only located in the active site of CI but could also impact local protein folding when carbonylated. This irreversible modification has been considered a biomarker for oxidative stress-induced cellular damage and may be a future therapeutic target for the prevention of epileptogenesis.
doi : 10.1016/j.freeradbiomed.2011.10.076
53 New and Improved Immuno-Spin Trapping Methods for the Detection of DNA Radicals Fiona A. Summers1, Ronald P. Mason1, and Marilyn Ehrenshaft1 1 NIEHS Reactive oxygen species have been implicated in DNA damage induced by certain drugs, environmental hazards and endogenous cellular processes. Unrepaired DNA damage can lead to cell death, cellular dysfunction and cancer. Although DNA radicals can be studied by electron spin resonance (ESR) using in vitro oxidizing systems, ESR is unable to detect DNA radical formation within an intact cell. We have developed a method to detect DNA radicals using immuno-spin trapping with the spin trap
SFRBM 2011
S31
doi : 10.1016/j.freeradbiomed.2011.10.071
48 Cell-Based Analysis of Lipid Peroxidation and Lipid Peroxidation-Derived Protein Modifications Using Fluorescence Microscopy and High Content Imaging Bhaskar S Mandavilli1, Robert J Aggeler1, Aleksey Rukavishnikov1, Chad Pickens1, Upinder Singh1, Hee Chol Kang1, Kyle Gee1, Brian Agnew1, and Michael S Janes1 1 Life Technologies Corporation’ Eugene, OR, USA Oxidative stress plays an important role in the progression of several diseases including inflammation, atherosclerosis, aging and age-related degenerative disorders. Reactive oxygen species damage membrane bound lipids including unsaturated fatty acids like linoleic acid and arachidonic acid to form lipid electrophiles, which can rapidly react with proteins and DNA to form adducts. Cell-based measurements of lipid peroxidation and protein carbonylation by traditional fluorescence microscopy and high content imaging provide a powerful platform to quantitate lipid peroxidation in cells and also to monitor spatial distribution of damage caused by lipid peroxides. Here, we used two different approaches to measure lipid peroxidation in cells. 1) a ratiometric determination of lipid peroxidation in live cells using a fluorescent probe which is incorporated into cellular membranes and emits at 590 nm. Oxidation of the dye resulted in a fluorescence shift to a peak emission of 510 nm. the ratio of 590/510 nm emissions can be quantitated by traditional fluorescence microscopy and high content imaging, 2) in a click chemistry-based approach, alkyneor azide-modified unsaturated fatty acid analogs like linoleic acid or arachidonic acid were incorporated into the cellular membranes and the products resulting from oxidation, like 4-hydroxy-2nonenal (HNE) and 9, 12-dioxo-10(E) dodecenoic acid (DODE) can readily modify DNA or proteins. the modified proteins are then detected by a copper-catalyzed click reaction using fluorescent alkynes or azides. Using these approaches, we measured lipid peroxidation caused by oxidants like tert-butyl hydroperoxide (TBHP), cumene hydroperoxide (CH), menadione, hemin and lipolysaccharide (LPS) in bovine pulmonary endothelial cells and murine macrophage-like cells. the oxidants produced 2-3 fold increases in lipid peroxidation and protein modifications when compared to controls. the lipid peroxidation and the lipid peroxidederived protein modifications were successfully inhibited by using antioxidants like α-tocopherol and mixed tocopherols. the two strategies described here to measure lipid peroxidation and the derived protein modifications provide powerful tools to measure oxidative stress in cells.
doi : 10.1016/j.freeradbiomed.2011.10.072
S30
49 Inactivation of α-Ketoglutarate Dehydrogenase by 4Hydroxy-2-nonenal; Modification and Metabolic Fate of the Cofactor Lipoic Acid Aaron L. McLain1,2, Kenneth M. Humphries1,2, Daniel Stofan1, Michael T. Kinter1, and Luke I. Szweda1,2 1 Free Radical Biology and Aging Program, Oklahoma Medical 2 Research Foundation, Department of Biochemistry, University of Oklahoma Health Sciences Center The mitochondrial electron transport chain is a source of oxygen derived free radicals and pro-oxidants. Polyunsaturated fatty acids present within the mitochondrial membrane are in close proximity and are highly susceptible to oxidation. Lipid peroxidation can disrupt mitochondrial function by compromising membrane integrity and through the formation of reactive aldehydes, alkenals, and hydroxyalkenals. We previously demonstrated that the lipid peroxidation product 4-hydroxy-2nonenal (HNE) readily inactivates α-ketoglutarate dehydrogenase upon modification of the covalently bound cofactor lipoic acid. This results in reductions in the rates of NADH production and oxidative phosphorylation. Results of the present study provide evidence that, of the various lipid peroxidation products, hydroxyalkenals are the most potent inactivators of αketoglutarate dehydrogenase. Because HNE is the most abundant hydroxyalkenal formed during lipid peroxidation, antibodies were prepared that are specific to the HNE-lipoic acid adduct, exhibiting no cross reactivity to adducts formed between lipoic acid and structurally related products. These antibodies were used to demonstrate modification of lipoic acid on the E2 subunit of α-ketoglutarate dehydrogenase upon treatment of mitochondria with HNE. Once formed, the HNE-lipoic acid adduct disappeared over time while the level of the E2 subunit remained unchanged. Evidence indicates that the lipoic acid-HNE adduct is removed from the protein as the first step in a repair process.
doi : 10.1016/j.freeradbiomed.2011.10.073
50 4-Hydroxy-2-Nonenal Mediates Aifm2 Release From Mitochondria: An Insight Into the Mechanism of Oxidative Stress Mediated Retrograde Signaling Sumitra Miriyala1, Chadinee Thipakkorn2, Yong Xu1, Teresa Noel1, Craig Vander Kooi1, Ines Batinic-Haberle3, Virapong Prachayasittikul2, and Daret St Clair1 1 2 3 University of Kentucky, Mahidol University, Duke University Mitochondria, in addition to generation of ATP and production of ROS, have a critical role in signaling events leading to apoptosis or survival under normal and pathophysiological conditions. However, it is unknown how ROS generated in mitochondria signals the communication between mitochondria and the nucleus under life and death conditions. Here, we report the identification of the mitochondrion-associated apoptotic protein 2 (AIFm2), a p53 target gene, as the messenger for ROS-mediated retrograde signaling under stress conditions. AIFm2 is a flavoprotein of relative molecular mass 41,000, which possesses NAD(P)H oxidase activity and catalyzes NAD(P)H dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. in vivo, treatment with Doxorubicin (Dox), a ROS generating chemotherapeutic drug, significantly enhanced the levels of 4-hydroxy-2-nonenal (4HNE) and AIFm2 protein in cardiac tissues. Using immunoprecipitation and immunofluorescence we found that resting AIFm2 localized to mitochondria, whereas HNE-adducted AIFm2 localized to nucleus suggesting that HNE modification activates the shuttling of AIFm2. When purified AIFm2 was exposed to 4HNE in vitro,
SFRBM 2011
4HNE selectively modified His174 and Cys187 of AIFm2 assessed by mass spectroscopic analysis. Lentivirus expressed wild-type and C187T mutant AIFm2 migrated from mitochondria to nucleus upon exposure to Dox which is prevented by addition of the MnSOD mimetic whereas the H174R mutant protein remained in the mitochondria after exposure to Dox. the HNE-adducted AIFm2 exhibited strong DNA binding affinity whereas the H174R mutant demonstrated no DNA binding capability. the results demonstrate that AIFm2 is a redox-sensitive protein and that its translocation is controlled by mitochondrial ROS, which may explain the apoptosis function associated with AIFm2 as a p53 target gene.
do i : 10.1016/j.freeradbiomed.2011.10.074
51 Structural Characterization of Oxidation Susceptible Cysteines: a Step Towards Understanding Hydrogen Peroxide Mediated Signaling Santhosh Kannan ` Venkatesan1, Boopathy Rathanam1, and Lakshmi Venkatachalam1 1 Department of Biotechnology, Bharathiar University, Coimbatore, India Protein thiols particularly redox regulatory cys are the primary and direct targets of H2O2 oxidation which in turn regulate H2O2 mediated signaling. the susceptibility of a cys is influenced by its physico-chemical parameters which are in turn governed by their site of distribution. Neither structural nor sequence determinants have been reported for H2O2 oxidizable cys. Hence, spatial and structural restraints that influence the oxidation of cys by H2O2 is a pre-requisite for the identification of target proteins. An attempt has been made to study the structural parameters that distinguish the susceptible cys from that of non-susceptible ones. A database of known H2O2 oxidizable (structural protein and six different classes of enzyme) proteins was generated. It consists of 64 susceptible cys residues and 70 non-susceptible ones. the parameters considered for identification of H2O2 susceptible cys were i) the pKa of target cys ii) distance to the adjacent cys S (S1) iii) solvent accessibility of S1 and iv) distance to the nearby acidic and basic amino acids. The pKa of most susceptible cys was found to be less than 8.5, while that of non-susceptible cys was above 9. When the pKa of susceptible cys is above the cut-off range of 8.5, the deprotonation of such target cys was achieved by S1 or by the adjacent acidic amino acid. They help in its maintenance as thiolate anion. the other susceptible cys residues whose pKa value is lower than 8.5 was observed only with oxido-reductases enzymes. the second determinant is the distance of S1 to the target cys. If it is at or below 6Å, it normally favored the target cys to its anionic form. a distance below 3Å resulted in permanent disulphide linkage thereby reducing the susceptibility to H2O2. So the pKa of target cys and distance of S1 to target cys were the major discriminating parameters that differentiate susceptible cys from non-susceptible ones. There were exceptions too, which were compensated by the increased solvent accessible area of the S1 and its proximity to acidic aminoacid. the validity of these parameters and their applicability were cross checked with two proteins which are known targets of H2O2. These parameters identified will help to develop an algorithm to predict target proteins for H2O2 signaling.
doi : 10.1016/j.freeradbiomed.2011.10.075
52 Carbonylation and Inhibition of Mitochondrial Complex I during Kainate-Induced Epileptogenesis Kristen R Ryan1, Li-Ping Liang1, Donald S Backos1, Philip Reigan1, and Manisha Patel1 1 University of Colorado Denver, Department of Pharmaceutical Sciences Nearly one percent of the population suffers from epilepsy and acquired epilepsies such as temporal lobe epilepsy (TLE) resulting from head trauma, chemical exposure or infections. Like many progressive disorders, mitochondrial dysfunction and oxidative stress are linked to the development of TLE. a single injection of kainate, a glutamatergic agonist, results in acute excitotoxicity and chronic development of spontaneous seizures via a process termed epileptogenesis. We have previously shown increased mitochondrial-specific reactive oxygen species (ROS) production, mitochondrial DNA damage and decreases in mitochondrial redox status in the hippocampus of a rat kainate model of epileptogenesis (Jarrett S et al 2008; Liang L et al 2006). Increasing evidence suggests that ROS-induced protein posttranslational modifications are a contributing factor to mitochondrial dysfunction. We hypothesize that Complex I (CI) dysfunction may be a major cause of mitochondrial dysfunction and contribute to epileptogenesis. the goal of this study was to determine if oxidative modification of the 75kDa subunit (Ndufs1) of CI occurs in the rat hippocampus during epileptogenesis and whether this correlates with disease progression. Rats were injected with a single high dose of kainate or vehicle and monitored by video for seizure activity for 6 weeks. Mitochondrial CI activity was significantly decreased acutely (8h-2d) and chronically (6wk) after kainate injection; whereas ATP levels were persistently decreased throughout the 6 week period. Evidence for CI modifications was measured acutely after kainate administration, days after kainate prior to development of epilepsy (i.e. latent period) and during the chronic stages of epilepsy. CI was immunoprecipitated (Mitosciences, OR) from hippocampal tissue of vehicle and kainate treated rats and subunits were separated by SDS-PAGE. Ndufs1 modification was identified with an Agilent 6510 QTOF LC/MS system and MS/MS peptide data was dually analyzed with Mascot 2.2 (Matrix Science) and SpectraMill (Agilent). a correlation was observed between total mitochondrial carbonylation, specific Ndufs1 carbonylation at Arginine 76 (Arg76), and inhibition of CI activity throughout the course of epileptogenesis. Molecular modeling (Accelrys Discovery Studio 3.0) studies revealed that Arg76 is not only located in the active site of CI but could also impact local protein folding when carbonylated. This irreversible modification has been considered a biomarker for oxidative stress-induced cellular damage and may be a future therapeutic target for the prevention of epileptogenesis.
doi : 10.1016/j.freeradbiomed.2011.10.076
53 New and Improved Immuno-Spin Trapping Methods for the Detection of DNA Radicals Fiona A. Summers1, Ronald P. Mason1, and Marilyn Ehrenshaft1 1 NIEHS Reactive oxygen species have been implicated in DNA damage induced by certain drugs, environmental hazards and endogenous cellular processes. Unrepaired DNA damage can lead to cell death, cellular dysfunction and cancer. Although DNA radicals can be studied by electron spin resonance (ESR) using in vitro oxidizing systems, ESR is unable to detect DNA radical formation within an intact cell. We have developed a method to detect DNA radicals using immuno-spin trapping with the spin trap
SFRBM 2011
S31