Immuno-spin Trapping Research Articles

A reciprocal relationship between mitochondria and lipid peroxidation determines the chondrocyte intracellular redox environment

In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.

The oxidation of fenamic acid NSAIDs by neutrophil myeloperoxidase produces toxic reactive metabolites that induce leukemic cell death

Fenamic acids are a group of non-steroidal anti-inflammatory drugs (NSAIDs) that are among the most common drugs prescribed globally. However, they have been associated with many adverse effects, such as agranulocytosis, neutropenia, hepatotoxicity, and nephrotoxicity. The interactions between peroxidase enzymes and fenamic acid-like NSAIDs cause the formation of reactive species, potentially involved in side effects. The aim of this study was to investigate the neutrophil myeloperoxidase (MPO)-mediated bioactivation of fenamic acids based on N-phenylanthranilic acid (NPA) and its four drug analogues: flufenamic acid (FFA), mefenamic acid (MFA), meclofenamic acid (MCFA), and tolfenamic acid (TFA). We hypothesized that the enzymatic oxidation of fenamic acids by MPO/hydrogen peroxide (H2O2) would produce reactive metabolites, cause oxidative damage and induce cytotoxicity. We utilized UV–Vis spectrophotometry, liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic spin resonance (EPR) spectroscopy using purified MPO from human neutrophils. In addition, in vitro studies were performed with MPO-containing human promyelocytic leukemia (HL-60) cells for cytotoxicity and immuno-spin trapping to detect protein-free radicals. UV–Vis spectrophotometry revealed that MPO oxidized the fenamic acids. LC-MS analyses revealed the formation of dimers, hydroxylated, and quinoneimine species, and glutathione (GSH) conjugates. EPR spin trapping with DMPO using GSH revealed that fenamic acids produced glutathionyl radicals in a concentration-dependent manner. We also detected the formation of protein-free radicals in HL-60 cells, which correlated with cytotoxicity. Despite the minor structural differences between the fenamic acids, there were variations in their oxidation potential. These findings revealed a correlation between pro-oxidant metabolite reactivity and cytotoxicity caused by fenamic acid NSAIDs.

Temporary alteration of neuronal network communication is a protective response to redox imbalance that requires GPI-anchored prion protein

Cellular prion protein (PrPC) protects neurons against oxidative stress damage. This role is lost upon its misfolding into insoluble prions in prion diseases, and correlated with cytoskeletal breakdown and neurophysiological deficits. Here we used mouse neuronal models to assess how PrPC protects the neuronal cytoskeleton, and its role in network communication, from oxidative stress damage. Oxidative stress was induced extrinsically by potassium superoxide (KO2) or intrinsically by Mito-Paraquat (MtPQ), targeting the mitochondria. In mouse neural lineage cells, KO2 was damaging to the cytoskeleton, with cells lacking PrPC (PrP-/-) damaged more than wild-type (WT) cells. In hippocampal slices, KO2 acutely inhibited neuronal communication in WT controls without damaging the cytoskeleton. This inhibition was not observed in PrP-/- slices. Neuronal communication and the cytoskeleton of PrP-/- slices became progressively disrupted and degenerated post-recovery, whereas the dysfunction in WT slices recovered in 5 days. This suggests that the acute inhibition of neuronal activity in WT slices in response to KO2 was a neuroprotective role of PrPC, which PrP-/- slices lacked. Heterozygous expression of PrPC was sufficient for this neuroprotection. Further, hippocampal slices from mice expressing PrPC without its GPI anchor (PrPGPI-/-) displayed acute inhibition of neuronal activity by KO2. However, they failed to restore normal activity and cytoskeletal formation post-recovery. This suggests that PrPC facilitates the depressive response to KO2 and its GPI anchoring is required to restore KO2-induced damages. Immuno spin-trapping showed increased radicals formed on the filamentous actin of PrP-/- and PrPGPI-/- slices, but not WT and PrP+/- slices, post-recovery suggesting ongoing dysregulation of redox balance in the slices lacking GPI-anchored PrPC. The MtPQ treatment of hippocampal slices temporarily inhibited neuronal communication independent of PrPC expression. Overall, GPI-anchored PrPC alters synapses and neurotransmission to protect and repair the neuronal cytoskeleton, and neuronal communication, from extrinsically induced oxidative stress damages.

Detection and characterization of free oxygen radicals induced protein adduct formation in differentiating macrophages

Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.

In vivo Detection of Macromolecule Free Radicals in Mouse Sepsis-Associated Encephalopathy Using a New MRI and Immunospin Trapping Strategy.

IntroductionFree radicals in oxidative stress are known to play a pathogenic role in sepsis. A major clinical challenge associated with sepsis is sepsis-associated encephalopathy (SAE). The rapid increase of free radicals in the brain promotes SAE progression. Here, macromolecule free radicals in the mouse brain were uniquely detected by immunospin trapping (IST) and magnetic resonance imaging (MRI).MethodsThe new strategy uses spin trapping agent DEPMPO-biotin to capture macromolecule free radicals in lesions and form biotin-DEPMPO-radical adducts. Then, a targeting MRI probe, avidin-BSA@Gd-ESIO, was used to detect the radical adducts through the highly specific binding of avidin and biotin. The avidin-BSA@Gd-ESIO probe was synthesized and systematically characterized. The detection capability of the new strategy was evaluated in vitro and in vivo using a confocal microscope and a 7T MRI, respectively.ResultsIn reactive oxygen species (ROS)–induced microglial cells, the accumulation of the avidin-BSA@Gd-ESIO probe in the DEPMPO-biotin-treated group was significantly higher than that of control groups. In vivo MRI T1 signal intensities were significantly higher within the hippocampus, striatum, and medial cortex of the brain in mice with a mild or severe degree of sepsis compared with the sham control group. Histological analysis validated that the distribution of the avidin-BSA@Gd-ESIO probe in brain tissue slices was consistent with the MRI images. The fluorescence signals of ROS and avidin-BSA@Gd-ESIO probe were overlapped and visualized using immunofluorescent staining. By evaluating the T1 signal changes over time in different areas of the brain, we estimated the optimal MRI detection time to be 30 minutes after the probe administration.DiscussionThis method can be applied specifically to assess the level of macromolecular free radicals in vivo in a simple and stable manner, providing a pathway for a more comprehensive understanding of the role of free radicals in SAE.

Extracellular biomolecular free radical formation during injury

ObjectiveDetermine if oxidative damage increases in articular cartilage as a result of injury and matrix failure and whether modulation of the local redox environment influences this damage.Osteoarthritis is an age associated disease with no current disease modifying approaches available. Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical to early degeneration, but these mechanisms have not been described in intact tissue. To assess free radical production as a result of traumatic injury, we measured biomolecular free radical generation via immuno-spin trapping (IST) of protein/proteoglycan/lipid free radicals after a 2 J/cm2 impact to swine articular cartilage explants. This technique allows visualization of free radical formation upon a wide variety of molecules using formalin-fixed, paraffin-embedded approaches. Scoring of extracellular staining by trained, blinded scorers demonstrated significant increases with impact injury, particularly at sites of cartilage cracking. Increases remain in the absence of live chondrocytes but are diminished; thus, they appear to be a cell-dependent and -independent feature of injury. We then modulated the extracellular environment with a pulse of heparin to demonstrate the responsiveness of the IST signal to changes in cartilage biology. Addition of heparin caused a distinct change in the distribution of protein/lipid free radicals at sites of failure alongside a variety of pertinent redox changes related to osteoarthritis. This study directly confirms the production of biomolecular free radicals from articular trauma, providing a rigorous characterization of their formation by injury.

Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1–10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Immuno-Spin Trapping Method for the Analysis of S-Nitrosylated Proteins.

The dynamic and unstable nature of protein nitrosothiols (PSNOs) derived from complex biological matrices (like cell lysates) make them unsuitable for proteomic/biochemical analysis in vitro. In an attempt to increase the stability of cell-derived PSNOs, scientists have devised methods to derivatize thiols undergoing nitrosylation, with a suitable molecule, to yield a stable adduct that could easily be detected using appropriate antibodies. The Biotin Switch Assay (BTSA) is currently the most widely used method for tagging PSNOs; however, the error-prone and cumbersome nature of the BTSA protocol prompted the development of alternative mechanisms of tagging cell-derived PSNOs. One such method is the immuno-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which effectively overcomes the shortcomings of the BTSA and proves to be a promising alternative. Here we describe the protocol for DMPO-based PSNO labeling and subsequent proteomic analysis by western blotting with an anti-DMPO antibody. © 2021 Wiley Periodicals LLC. Basic Protocol: Labeling of cell-derived PSNOs by DMPO-based immuno-spin trapping and their subsequent analysis by immunostaining.

Synthesis and evaluation of cyclic nitrone derivatives as potential anti-cancer agents

Nitrones have been found to exhibit attractive biological values as immuno spin trapping agents. However, successful clinical cases of nitrone therapeutics are still lacking. Herein we report the synthesis and antiproliferative activity of a series of structurally diverse nitrone derivatives against a panel of 5 cancer cell lines, based on which indole- and pyrrole-fused were further evaluated by analogue preparation and in-vitro screening. Analogues with moderate to good potency were identified. This study shows the promise for further pursuit of nitrone-type small molecules in chemotherapy.

Immuno-spin Trapping of Copper Nanoparticles for Beta-amyloid Protein Radical Formation: Implication of Protein Radical Formation in Alzheimer's Disease

Immuno-spin Trapping of Copper Nanoparticles for Beta-amyloid Protein Radical Formation: Implication of Protein Radical Formation in Alzheimer's Disease

Using MRI to measure in vivo free radical production and perfusion dynamics in a mouse model of elevated oxidative stress and neurogenic atrophy

Mitochondrial dysfunction, reactive oxygen species (ROS) and oxidative damage have been implicated to play a causative role in age-related skeletal muscle atrophy and weakness (i.e. sarcopenia). Mice lacking the superoxide scavenger CuZnSOD (Sod1−/−) exhibit high levels of oxygen-derived radicals and oxidative damage, associated with neuronal and muscular phenotypes consistent with sarcopenia. We used magnetic resonance imaging (MRI) technology combined with immunospin-trapping (IST) to measure in vivo free radical levels in skeletal muscle from wildtype, Sod1−/− and SynTgSod1−/− mice, a mouse model generated using targeted expression of the human Sod1 transgene specifically in neuronal tissues to determine the impact of motor neuron degeneration in muscle atrophy. By combining the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) and molecular MRI (mMRI), we monitored the level of free radicals in mouse hindlimb muscle. The level of membrane-bound macromolecular radicals in the quadriceps muscle was elevated by ~3-fold in Sod1−/− mice, but normalized to wildtype levels in SynTgSod1−/− rescue mice. Skeletal muscle mass was reduced by ~25–30% in Sod1−/− mice, but fully reversed in muscle from SynTgSod1−/− mice. Using perfusion MRI we also measured the dynamics of blood flow within mouse hindlimb. Relative muscle blood flow in Sod1−/− is decreased to ~50% of wildtype and remained low in the SynTgSod1−/− mice. Our findings are significant in that we have shown for the first time that in vivo free radical production in skeletal muscle is directly correlated to muscle atrophy in an experimental model of oxidative stress. Neuron-specific expression of CuZnSOD reverses the in vivo free radical production in skeletal muscle in the Sod1−/− mouse model and prevents muscle atrophy. These results further support the feasibility of using in vivo assessments of redox status in the progression of a pathological process such as sarcopenia. This approach can also be valuable for evaluating responses to pharmacologic interventions.

Characterization of Protein Radicals in Arabidopsis.

Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Reactive oxygen species (ROS) exhibit different spatial and temporal distributions as well as concentrations in- and outside the cell, thereby functioning as signaling or pathogen-destroying molecules. Especially the ROS H2O2 is important for the patho/physiological status of an organism. Electrochemistry (EM) and electron spin resonance (ESR)-based techniques allow quantification of H2O2 in artificial and living systems, coping a concentration range from low nM up to mM. Working electrodes for EM are optimized by diverse modifications and, additionally, redox mediators are used. Ultramicroelectrodes allow scanning of single cells to spatially resolve and quantify extracellular H2O2 in real-time. With ESR spectroscopy, •O2¯, but not H2O2, can be directly determined by spin probes in- and outside of cells in suspensions. Monitoring H2O2 requires formation of intermediate radicals, detectable with spin probes. Low μM [H2O2] can thus be assessed specifically. Using suitable spin traps, in-vivo ESR and immuno-spin trapping can visualize different radicals at their respective production sites in small animals, organs and tissues. Here, the redox reaction cascades may interfere with cell metabolism. Optimization of all methods established for H2O2 determination would be favorable to finally combine them for mutual validation. Thus, a deeper insight into cellular ROS metabolism can be obtained.

294 - MRI imaging for in vivo free radicals and bioenergetics in mouse skeletal muscle

Background Accumulation of mitochondrial dysfunction, reactive oxygen species (ROS) and oxidative damage play a causal role in skeletal muscle atrophy and contractile dysfunction. Magnetic resonance imaging (MRI) technology is an excellent tool to assess redox imbalance within tissue and to measure dynamics of blood flow and oxygenation in skeletal muscle in vivo real time. Methods We used wildtype mice, mice lacking the cytosolic superoxide scavenger CuZn-superoxide dismutase (CnZnSOD, Sod1-/- mice), and a mouse model engineered to express Sod1 selectively in neuronal tissue under the control of synapsin 1 promoter on an Sod1-/- genetic background (neuronal SynTgSod1-/- rescue mice) to test the role of neurogenic oxidative stress in skeletal muscle redox status. By combining molecular magnetic resonance imaging (mMRI) and immunospin-trapping technologies using DMPO, we monitored the levels of free radicals in vivo real time. For functional MRI, we placed an electrode directly on gastrocnemius. A 15-min exercise protocol was achieved by electrical stimulations (2 mA, 0.5 Hz, 0.2 ms pulse duration). We obtained relative changes of blood oxygenation level dependent-signal intensity (BOLD-SI) of exercising skeletal muscle using a 7.0T MRI system. Results In vivo free radical levels in lower limb muscles were elevated by ~3 fold in Sod1-/- mice but normalized to wildtype level in SynTgSod1-/- mice. Skeletal muscle mass was significantly reduced in Sod1-/-, but fully reversed in SynTgSod1-/-. Relative muscle oxygenation was decreased Sod1-/- mice and remained reduced in SynTgSod1-/- . Similarly, relative muscle blood flow in Sod1-/- is decreased to ~50% of wildtype and remained low in SynTgSod1-/- mice. Conclusion We demonstrate that free radical levels can be assessed by MRI coupled with immunospin trapping in skeletal muscle. Our data also indicates an inverse relationship between muscle mass and in vivo free radicals in muscle. Neuron-specific expression of CuZnSOD prevented muscle atrophy independent of perfusion rate or oxgen tension in muscle.

326 - In vivo detection of free radicals in rats with LPS-induced encephalopathy using molecular-targeted MRI and treatment with the anti-inflammatory agent, OKN-007

It is known that long-term neuroinflammation is correlated with vascular and cognitive impairment. Sepsis is a severe acute systemic inflammatory reaction to circulating bacterial toxins, and is a serious and potentially fatal illness, that can result in multi-organ dysfunction and failure. In this study, we used a rat model for septic encephalopathy to induce inflammation and assess in vivo levels of free radicals in non-treated and OKN-007-treated rats. In vivo free radical levels were assessed using immuno-spin trapping coupled with molecular-targeted MRI (mtMRI). Lipopolysaccharide (LPS) was used to induce encephalopathy. Rats (male; n=10 anti-DMPO (5,5-dimethyl-1-pyrroline-N-oxide)-albumin-Gd-DTPA-biotin (anti-DMPO-probe) (n=5 saline-treated; n=5 LPS-exposed; 24 hours), and n=10 anti-DMPO-albumin-Gd-DTPA-biotin (anti-DMPO-probe) (n=5 saline-treated; n=5 LPS-exposed; 1 week), were anesthetized with isoflurane for LPS or saline administration (i.p.), and for MRI scans. A targeted contrast agent probe for DMPO trapped free radicals was used to obtain trapped macromolecular free radical images. Multiple brain region MR image slices were obtained. Rat brains were imaged at pre-contrast and at several intervals post-contrast agent injection. T1-weighted images were also obtained. Pixel-by-pixel relaxation maps were reconstructed from a series of T1-weighted images using a nonlinear two-parameter fitting procedure. The T1 value of a specified region-of-interest (ROI) was computed from all the pixels in the identified ROIs. mtMRI was able to demonstrate increased free radical levels in LPS-exposed rat brains at both 24 hours and 1 week post-LPS administration, compared to saline-treated controls, and decreased free radical levels 1 week after OKN-007 treatment. Data indicates the feasibility of conducting in vivo mtMR imaging for free radical levels in a rat model for encephalopathy begfore and after treatment with the anti-inflammatory agent, OKN-007. Here we used mtMRI to show for the first time non-invasive in vivo detection of free radical levels associated with endotoxia-induced encephalopathy, and the effect of a therapeutic intervention.

Organic radical imaging in plants: Focus on protein radicals

Biomolecule (lipid and protein) oxidation products formed in plant cells exposed to photooxidative stress play a crucial role in the retrograde signaling and oxidative damage. The oxidation of biomolecules initiated by reactive oxygen species is associated with formation of organic (alkyl, peroxyl and alkoxyl) radicals. Currently, there is no selective and sensitive technique available for the detection of organic radicals in plant cells. Here, based on the analogy with animal cells, immuno-spin trapping using spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to image organic radicals in Arabidopsis leaves exposed to high light. Using antibody raised against the DMPO nitrone adduct conjugated with the fluorescein isothiocyanate, organic radicals were imaged by confocal laser scanning microscopy. Organic radicals are formed predominantly in the chloroplasts located at the periphery of the cells and distributed uniformly throughout the grana stack. Characterization of protein radicals by standard immunological techniques using anti-DMPO antibody shows protein bands with apparent molecular weights of 32 and 34 kDa assigned to D1 and D2 proteins and two protein bands below the D1/D2 band with apparent molecular weights of 23 and 18 kDa and four protein bands above the D1/D2 band with apparent molecular weights of 41, 43, 55 and 68 kDa. In summary, imaging of organic radicals by immuno-spin trapping represents selective and sensitive technique for the detection of organic radicals that might help to clarify mechanistic aspects on the role of organic radicals in the retrograde signaling and oxidative damage in plant cell.

Immuno-Spin Trapping-Based Detection of Oxidative Modifications in Cardiomyocytes and Coronary Endothelium in the Progression of Heart Failure in Tgαq*44 Mice.

Recent studies suggest both beneficial and detrimental role of increased reactive oxygen species and oxidative stress in heart failure (HF). However, it is not clear at which stage oxidative stress and oxidative modifications occur in the endothelium in relation to cardiomyocytes in non-ischemic HF. Furthermore, most methods used to date to study oxidative stress are either non-specific or require tissue homogenization. In this study, we used immuno-spin trapping (IST) technique with fluorescent microscopy-based detection of DMPO nitrone adducts to localize and quantify oxidative modifications of the hearts from Tgαq*44 mice; a murine model of HF driven by cardiomyocyte-specific overexpression of Gαq* protein. Tgαq*44 mice and age-matched FVB controls at early, transition, and late stages of HF progression were injected with DMPO in vivo and analyzed ex vivo for DMPO nitrone adducts signals. Progressive oxidative modifications in cardiomyocytes, as evidenced by the elevation of DMPO nitrone adducts, were detected in hearts from 10- to 16-month-old, but not in 8-month-old Tgαq*44 mice, as compared with age-matched FVB mice. The DMPO nitrone adducts were detected in left and right ventricle, septum, and papillary muscle. Surprisingly, significant elevation of DMPO nitrone adducts was also present in the coronary endothelium both in large arteries and in microcirculation simultaneously, as in cardiomyocytes, starting from 10-month-old Tgαq*44 mice. On the other hand, superoxide production in heart homogenates was elevated already in 6-month-old Tgαq*44 mice and progressively increased to high levels in 14-month-old Tgαq*44 mice, while the enzymatic activity of catalase, glutathione reductase, and glutathione peroxidase was all elevated as early as in 4-month-old Tgαq*44 mice and stayed at a similar level in 14-month-old Tgαq*44. In summary, this study demonstrates that IST represents a unique method that allows to quantify oxidative modifications in cardiomyocytes and coronary endothelium in the heart. In Tgαq*44 mice with slowly developing HF, driven by cardiomyocyte-specific overexpression of Gαq* protein, an increase in superoxide production, despite compensatory activation of antioxidative mechanisms, results in the development of oxidative modifications not only in cardiomyocytes but also in coronary endothelium, at the transition phase of HF, before the end-stage disease.

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

Exposure to (bi)sulfite (HSO3–) and sulfite (SO32–) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–), peroxymonosulfate (–O3SOO.), and especially the sulfate (SO4. –) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of protein radicals in a mouse model of human lung disease.

In Vivo and In Situ Detection of Macromolecular Free Radicals Using Immuno-Spin Trapping and Molecular Magnetic Resonance Imaging.

In vivo free radical imaging in preclinical models of disease has become a reality. Free radicals have traditionally been characterized by electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy coupled with spin trapping. The disadvantage of the ESR/EPR approach is that spin adducts are short-lived due to biological reductive and/or oxidative processes. Immuno-spin trapping (IST) involves the use of an antibody that recognizes macromolecular 5,5-dimethyl-pyrroline-N-oxide (DMPO) spin adducts (anti-DMPO antibody), regardless of the oxidative/reductive state of trapped radical adducts. Recent Advances: The IST approach has been extended to an in vivo application that combines IST with molecular magnetic resonance imaging (mMRI). This combined IST-mMRI approach involves the use of a spin-trapping agent, DMPO, to trap free radicals in disease models, and administration of an mMRI probe, an anti-DMPO probe, which combines an antibody against DMPO-radical adducts and an MRI contrast agent, resulting in targeted free radical adduct detection. The combined IST-mMRI approach has been used in several rodent disease models, including diabetes, amyotrophic lateral sclerosis (ALS), gliomas, and septic encephalopathy. The advantage of this approach is that heterogeneous levels of trapped free radicals can be detected directly in vivo and in situ to pin point where free radicals are formed in different tissues. The approach can also be used to assess therapeutic agents that are either free radical scavengers or generate free radicals. Smaller probe constructs and radical identification approaches are being considered. The focus of this review is on the different applications that have been studied, advantages and limitations, and future directions. Antioxid. Redox Signal. 28, 1404-1415.

87 - Induction of Beta amyloid Radical fFrmation through Immuno-spin trapping of Copper nano particles: Implication of Protein Radicals in Alzheimer's Disease

Alzheimer’s disease (AD) is a debilitating progressive neurodegenerative disease that usually starts slowly and worsens over time. Though AD etiology is believed to be multifactorial, there is considerable evidence showing the role of copper exposure in beta-amyloid (βA) aggregation and AD pathogenesis. Owing to the fact that βA aggregation takes place in the synaptic cleft where copper is found in high concentrations, copper exposure especially in the form of nano particles can be more detrimental. Though copper-induced oligomeric forms of βA accumulate and significantly damage the brain cells, inducing cognitive dysfunctions, the mechanism that initiates and promotes βA oligomerization remains unknown. We hypothesized protein radical formation as an initiating mechanism for βA oligomerization. Therefore, we used the highly sensitive immuno-spin trapping technique to investigate protein radical formation as a possible mechanism of βA oligomerization. Here, we have investigated copper nano particles induced βA radical formation and results clearly showed that both variants of nano particles (25 and 70 nm sizes) significantly produced βA radical in presence of hydrogen peroxide. To further validate copper nano particle-induced protein radical formation in biological context, we exposed IMR-32 hippocampal neuroblastoma cells expressing βA to both variants of copper nano particles and analyzed protein radical formation using confocal microscopy. Confocal microscopy results clearly showed protein radical formation in IMR-32 cells exposed to either variant of copper nano-particles in presence of hydrogen peroxide. Taken together, these results clearly suggest that both variants of copper nano particles induce βA radical formation which can lead to βA oligomerization, and therefore contributes to AD pathogenesis.